A new procedure for deconvolution of inter-/intramolecular intrinsic primary and α-secondary deuterium isotope effects from enzyme steady-state kinetic data

J. Am. Chem. Soc. 1999, 121, 5865-5880

5865

A New Procedure for Deconvolution of Inter-/Intramolecular Intrinsic

Primary and R-Secondary Deuterium Isotope Effects from Enzyme

Steady-State Kinetic Data

William S. McIntire,*^,†,‡E. Thomas Everhart,^§,John C. Craig,^§and Vladislav Kuusk^†,|

Contribution from the Molecular Biology DiVision, Department of Veterans Affairs Medical Center,

San Francisco, California 94121, and Department of Pharmaceutical Chemistry,

Department of Biochemistry and Biophysics, and Department of Anesthesia, UniVersity of California,

San Francisco, California 94143

ReceiVed December 7, 1998. ReVised Manuscript ReceiVed March 18, 1999

Abstract: The A₂B₂flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida oxidizes

4-methylphenol (p-cresol) to 4-hydroxybenzyl alcohol in a process requiring scission of an R-C-H bond with

concomitant reduction of covalently bound FAD in each A subunit. Values of k_cat/K were determined from

steady-state kinetic data for the reactions of PCMH with the following substrates: 4-methylphenol, 4-(²H₁)-

methylphenol, 4-(²H₂)methylphenol, and 4-(²H₃)methylphenol. A procedure was devised to extract the intrinsic

primary deuterium and intrinsic R-secondary deuterium kinetic isotope effects from these values of k_cat/K. The

primary effect, P, is 6.71 ( 0.08, and the secondary effect, S, is 1.013 ( 0.014. The magnitudes of these

effects are discussed in terms of an early or late transition state, hydrogen tunneling, coupled motion between

the leaving and remaining hydrogens of the methyl group, and a H^-expulsion mechanism versus a substrate

radical mechanism versus a covalent substrate-FAD intermediate mechanism. The reaction of 4-ethylphenol

with PCMH produces 4-vinylphenol and (-)-S-1-(4-hydroxyphenyl)ethanol (∼100% enantomeric excess). The

evidence indicates that these are formed from a common intermediate, presumably a p-quinone methide. From

the partition ratios for the formation of the alcohol and 4-vinylphenol from 4-ethylphenol, 4-(1′,1′-²H₂)-

ethylphenol, and 4-(2′,2′,2′-²H₃)ethylphenol, the primary isotope effect for conversion of the p-quinone (2′,2′,2′-

²H₃)methide to 4-(2′,2′-²H₂)vinylphenol was estimated to be about 2, and the R-secondary isotope effect for

conversion of p-quinone (1′-²H₁)methide to 1-(4-hydroxyphenyl)-(1′-²H₁)ethanol was found to be inverse

()0.83), as expected for sp²to sp³hybridization change at the R-carbon. Values of k_cat/K were determined for

4-ethylphenol, R,S-(()-4-(1′-²H₁)ethylphenol (abbreviated R,S-D), S-(-)-4-(1′-²H₁)ethylphenol (S-D), R-(+)-

4-(1′-²H₁)ethylphenol (R-D), and 4-(1′,1′-²H₂)ethylphenol (D2). The ^D2(k_cat/K) value was found to be 5.1-6.1,

the same as determined in an earlier study. Unexpectedly, the values for ^R,S-D(k_cat/K), ^S-D(k_cat/K), and

^R-D(k_cat/K) were all about the same (∼1.7), indicating that there is nearly an equal probability for pro-R or

pro-S C-H bond scission. An apparent flux ratio for the pro-S path/pro-R path was estimated to be 0.78 (

0.02. The same procedure devised to determine values for P and S for 4-methylphenol was used to determine

these values for the 4-ethylphenol reaction (commitment to catalysis ) 0); P ) 5.98 ( 0.12 and S ) 0.967

( 0.021. These values are essentially the same as those determined for 4-methylphenol. Thus, the chemical

mechanisms for both substrates are assumed to be similar.

Introduction

experimental work, of the past two decades have indicated that

interpretation of these effects are complicated by the possible

involvement of H/D tunneling and/or coupled motion of

secondary H/D with the breaking of the primary H/D-C bond.^1,2

However, these complexities have not diminished the interest

or importance of deuterium isotope effects.^2-4Thus, develop-

ment of new, fast, and convenient methods of extracting these

effects from kinetic data is of general interest. While working

with several enzymes that oxidize methyl groups of their

substrates, we pondered different methods for determining the

intrinsic primary and secondary effects for the transformations

of these groups.

When only the trideuteriomethyl substrate is compared with

unlabeled substrate, the true primary and R-secondary effects

are impossible to separate, since the measured isotope effect is

intramolecular in nature. The observed effect is the product of

primary, P, and secondary effects, S, KIE ) PS², since one

deuterium is removed and two secondary deuteriums remain.

The special nature of the methyl group allows for each H/D

Intrinsic primary and especially secondary deuterium isotope

effects have proven to be extremely useful in deciphering the

exact chemical mechanisms of action for numerous enzymes.^1-3

Revelations, not to mention a great deal of theoretical and

* Address correspondence to Dr. William S. McIntire, Molecular Biology

Division (151-S), Department of Veterans Affairs Medical Center, 4150

Clement St., San Francisco, CA 94121. Phone: (415) 387-1431. Fax: (415)

750-6959. E-mail: wsm@itsa.ucsf.edu.

^†Department of Veterans Affairs Medical Center and Department of

Biochemistry and Biophysics, University of California, San Francisco.

^‡Department of Anesthesia, University of California, San Francisco.

^§Department of Pharmaceutical Chemistry, University of California, San

Francisco.

Current address: Department of Psychiatry, University of California,

San Francisco.

^|Current address: OREAD, Inc., 3401P Hillview Ave., Palo Alto, CA

94304.

(1) Kurz, L. C.; Freiden, C. J. Am. Chem. Soc. 1980, 102, 4198-4203.

Kohen, A.; Klinman J. P. Acc. Chem Res. 1998, 31, 397-404.

(2) (a) Klinman, J. P. In Enzyme Mechanisms from Isotope Effects; Cook,

P. F., Ed.; CRC Press: Boca Raton, FL, 1991; pp 127-148. (b) Bahnson,

B. J.; Klinman, J. P. Methods Enzymol. 1995, 249, 373-397.

(3) Fisher, H. F.; Saha, S. K. Biochemistry 1996, 35, 83-88.

(4) Enzyme Mechanisms from Isotope Effects; Cook, P. F., Ed.; CRC

Press: Boca Raton, FL, 1991.

Published on Web 06/09/1999

5866 J. Am. Chem. Soc., Vol. 121, No. 25, 1999

McIntire et al.

position to be spatially equivalent, assuming that there is little

or no isotope effect for positioning H or D for bond scission.

Under some circumstances, it might be possible to extract the

intrinsic effects from steady-state kinetic constants for the

unlabeled (D0), the mono- (D1), the di- (D2), and the trideu-

terated (D3) substrates.

Scheme 1

For D1, D2, and D3, steady-state constants k_cat/K_mand k_cat

will be affected by intra- and/or intermolecular isotope effects.

With D1, some molecules will undergo scission of a C-H bond,

whereas others will undergo a C-D bond scission. Therefore,

the effect on the kinetic constants will be intermolecular

(competition between two molecules) in nature. In contrast, for

D2, some molecules will undergo C-D scission and will

produce an SP intramolecular isotope effect, while other

molecules will undergo C-H bond cleavage, for an intramo-

lecular S²effect. In this case, the alteration in values of kinetic

parameters will result from inter- and intramolecular effects.

As mentioned earlier, the D3 rate constants will be influenced

by an intramolecular effect only.

Previously, it was demonstrated that 4-ethylphenol was

converted to optically active 1-(4-hydroxyphenyl)ethanol, which

was composed of 66% S-isomer and 34% R-isomer.¹⁶Using

an improved method described herein, it was demonstrated that

the alcohol product of 4-ethylphenol, in fact, is >97% S-isomer.

In another report, the intrinsic deuterium kinetic isotope effects

(KIEs) were determined for 4-methylphenol/4-(²H₃)methylphenol

(KIE ) 7.05) and for 4-ethylphenol/4-(1′,1′-²H₂)ethylphenol

(KIE ) 4.8-5.3).¹⁴However, because of the multiple deuterium

label in the R position, the isotope effects are the product of

the intrinsic primary and intrinsic R-secondary effects.

This special nature of the methyl group has been appreciated

by other research groups, and methyl intra-/intermolecular

effects have been measured for a number of enzymes.^5-8

However, in these cases, the isotope effects were derived by

analyzing the deuterium content of extracted material by mass

spectral analyses. While considering this approach, it occurred

to us that, under the right circumstances, it should be possible

to extract intrinsic primary and secondary deuterium isotope

effects directly from an analysis of steady-state kinetic data.

It was decided to test this notion using the A form of p-cresol

(4-methylphenol) methylhydroxylase [PCMH; p-cresol:(accep-

tor) oxidoreductase (methyl hydroxylating); EC 1.17.99.1] from

Pseudomonas putida N.C.I.M.B. 9869. PCMH is composed of

two flavoprotein subunits and two c-type cytochrome subunits.⁹

The enzyme belongs to a subclass of flavoproteins that have

FAD covalently bound at the 8R-carbon of the isoalloxazine

ring.^10-12The preferred substrate, 4-methylphenol (p-cresol),

is thought to be oxidized by bound FAD to p-quinone methide,

which is hydrated to give product, 4-hydroxybenzyl alcohol.

This alcohol subsequently can be converted to 4-hydroxyben-

zaldehyde by oxidized enzyme. PCMH can convert a variety

of other 4-alkylphenols to R-carbinols and, interestingly, to

4-vinylphenols. The R-carbinols derived from n-alkylphenols

are further oxidized by PCMH to the R-carbonyl derivatives.^13,15

PCMH presents a case that should be ideal for testing the method

for direct extraction of the isotope effects from steady-state

kinetic data, since it was found that ^D(k_cat/K_m) ) ^Dk₂for 4-(²H₃)-

methylphenol.¹⁴

The following compounds were synthesized with the aim of

determining the magnitudes of the intrinsic primary and

secondary deuterium isotope effects and the stereochemistry of

the reactions catalyzed by PCMH: R-, S-, and R,S-4-(1′-²H₁)-

ethylphenol; 4-(1′,1′-²H₂)ethylphenol; 4-(2′,2′,2′-²H₃)ethylphenol;

4-(²H₁)methylphenol, 4-(²H₂)methylphenol, and 4-(²H₃)-

methylphenol; and R-, S-, and R,S-1-(4-hydroxyphenyl)ethanol

and the corresponding 1′-²H-ethyl derivatives. This treatise

presents the results of studies carried out using these compounds.

It will be demonstrated that the true primary and secondary

intrinsic effects can be extracted from the values of the various

(k_cat/K_m)_H/Dconstants for 4-methylphenol. It will also be

demonstrated, with reasonable assumptions, that these effects

can also be derived from the values of (k_cat/K_m)_H/Dfor the

4-ethylphenol derivatives, which undergo oxidation of an

R-methylene carbon atom. Because the binding of 1-(4-

hydroxyphenyl)ethanol is slow and rate limiting, it was not

possible to determine the isotope effects from steady-state data.

Results

Intrinsic Primary and Secondary r-Deuterium Kinetic

Isotope Effects for the Oxidation of 4-Methylphenol. The

proposed mechanism for PCMH oxidation of 4-methylphenol

is presented in Scheme 1. PCMH converts 4-methylphenol to a

putative p-quinone methide by removal of the equivalent of two

protons and two electrons. The quinone methide is subsequently

attacked by water at its most nucleophilic center to produce an

alcohol. A similar oxidation of the alcohol by the enzyme yields

4-hydroxbenzaldehyde. Previously, a large isotope effect (7.03

( 0.41) on k_cat/K_4-MPwas found with 4-methylphenol (4-MP)

and 4-(²H₃)methylphenol as substrates. Stopped-flow kinetic

measurements confirmed that this is the intrinsic effect.¹⁴

However, this effect is the product of intrinsic primary and

R-secondary effects. With the goal of measuring these two

intrinsic effects, k_cat/K_4-MPvalues were determined from steady-

state kinetic assays done at a fixed concentration of PES (1.0

mM phenazine ethosulfate), with varying concentrations of

4-methylphenol (D0), 4-(²H₁)methylphenol (D1), 4-(²H₂)-

methylphenol (D2), or 4-(²H₃)methylphenol (D3). Earlier,¹⁴it

was found that the reaction of PCMH with PES and 4-meth-

ylphenol obeyed a ping-pong mechanism; thus, the slopes of

1/V vs 1/[4-methylphenol] plots at all [PES] are parallel and

will provide the true value for k_cat/K_4-MP(the slope of 1/V vs

(5) Hanzilik, R. P.; Hogberg, K.; Moon, J. P.; Judson, C. M. J. Am.

Chem. Soc. 1985, 107, 7164-7167.

(6) Hanzlik, R. P.; Schaefer, A. R.; Moon, J. B.; Judson, C. M. J. Am.

Chem. Soc. 1987, 109, 4926-4930.

(7) Jones, J. P.; Trager, W. F. J. Am. Chem. Soc. 1987, 109, 2171-

2173.

(8) Miller, V. P.; Tschirret-Guth, R. A.; Ortiz de Montellano, P. R. Arch.

Biochem. Biophys. 1995, 319, 333-340.

(9) McIntire, W. S.; Singer, T. P.; Smith, A. J.; Mathews, F. S.

Biochemistry 1986, 25, 5975-5981.

(10) McIntire, W. S.; Edmondson, D. E.; Hopper, D. J.; Singer, T. P.

Biochemistry 1981, 20, 3068-3075.

(11) Singer, T. P.; McIntire, W. S. Methods Enzymol. 1984, 106, 369-

378.

(12) Mewies, M.; McIntire, W. S.; Scrutton, N. S. Protein Sci. 1998, 7,

7-20.

(13) McIntire, W. S.; Hopper, D. J.; Singer, T. P. Biochem. J. 1985,

228, 325-335.

(14) McIntire, W. S.; Hopper, D. J.; Singer, T. P. Biochemistry 1987,

26, 4107-4117.

(15) McIntire, W. S.; Koerber, S. C.; Bohmont, C.; Singer, T. P. In

FlaVins and FlaVoproteins; Bray, R. C., Engel, P. C., Mayhew, S. G., Eds.;

Walter de Gruyter: Berlin and New York, 1984; pp 643-658.

(16) McIntire, W. S.; Hopper, D. J.; Craig, J. C.; Everhart, E. T.; Webster,

R. V.; Causer, M. J.; Singer, T. P. Biochem. J. 1984, 224, 617-621.

Isotope Effects and PCMH

J. Am. Chem. Soc., Vol. 121, No. 25, 1999 5867

Scheme 2

k_HDH/k_HDD) k_DHH/k_DHD) k_DDH/k_DDD; i.e., the primary effect

and secondary effects are independent (the first atom in the

subscript is the one removed from substrate). The rule is invalid

only when there is coupled motion between the removed and

remaining H/D atoms.²The analysis of the isotope effect could

also be complicated by tunneling. As will be discussed later, it

is unlikely that both tunneling and coupled motion are at work

in the reaction of PCMH. The last line in Scheme 2 represents

the reoxidation of reduced E (E′) by PES. Since the mechanism

is of a ping-pong type,¹⁴this portion of the mechanism is

uncoupled from the half-reaction involving 4-methylphenol, and

it is unaffected by the isotopic labeling of the substrate.

The equations for k_cat/K_4-MPfor D0, D1, D2 and D3 are given

below (eq 2a-d), where C ) k₂/k_-1is the forward commitment

to catalysis factor^15,18,19and X ) k₁k₂/k_-1) k₂/K_D. Also provided

are the same equations when C ) 0 (k_-1. k₂), which is the

case for the PCMH reaction with 4-methylphenol.¹⁴

1/[4-methylphenol] plots is K_4-MP/k_cat), regardless of the [PES].

The values of the parameters Q_X() k_cat/K_X) and K_Xwere

determined for X ) D0, D1, D2, or D3 by nonlinear regression

analysis¹³using eq 1.¹⁷

k_cat

X

k_cat[S]

(k_cat/K_X)[S]

Q_X[S]

)

) X

(2a)

(2b)

( )

V )

)

(1)

K

D0

1 + C

K_X+ [S] 1 + [S]/K_X1 + [S]/K_X

k_cat

X(3spC + 2s + p)

X(2s + p)

)

( )

3(1 + [s + p]C + spC²)

From the values of k_cat/K_X, three isotope effects on this parameter

were calculated. This series of experiments was done four times

(Table 1). For each set of experiments, different solutions of

D0, D1, D2, D3, and PCMH were used to minimize systematic

error. The ^D3(k_cat/K_4-MP) isotope effect (weighted average ) 6.63

( 0.08) is the same as determined earlier.¹⁴By direct inspection

of these values, it is not possible to discern the values of the

true intrinsic primary and R-secondary isotope effects.

K

D1

3

Xs(3s²pC + s + 2p)

3(1 + s²C + spC + s³pC²)

X(s²+ 2sp)

k_cat

)

(2c)

(2d)

( )

K

D2

3

Xs²p

k_cat

)

) Xs²p

( )

1 + s²pC

K

D3

Scheme 2 presents the model used to derive the equations

necessary to extract the intrinsic primary and secondary effects.

In the scheme, a substrate subscript represents one of the three

methyl hydrogens/deuteriums that can be removed; that is, for

D1, S_adenotes substrate that binds with one of the hydrogens

positioned to be removed, S_bdenotes substrate that binds with

the other hydrogen positioned to be removed, and S_cdenotes

substrate that is positioned for removal of deuterium. While

isotope effects on binding of substrate have been reported, it is

unlikely that the effect will be significant for a compound with

a labeled methyl group. Thus, it was assumed that there are no

isotope effects for substrate binding or release.¹⁸Therefore, for

D0, k_1a) k_1b) k_1c) k₁, k_2a) k_2b) k_2c) k₂, and k_3a) k_3b

) k_3c) k₃. Since k_-1> k₂for PCMH,¹⁴even if the methyl

group rotates when bound at the active site of PCMH (i.e., ES_a

a ES_ba ES_ca ES_a), the resulting equations are unchanged

(assuming no isotope effect for rotation). (This is not true if

(See the Experimental Procedures section for details of the

method used to derive the expressions in equation sets 2-5.)

With C > 0, it seemed that it would be possible to solve these

four equations for S (the intrinsic R-secondary effect) ) 1/s, P

(the intrinsic primary effect) ) 1/p, and C. Unfortunately, the

solutions for each of these parameters are transcendental

functions. Simulations (unpublished results) indicate that the

various k_cat/K_4-MPvalues would need to be measured accurately

and precisely to approximately 1 part in 10 000 in order to

determine the values of these parameters.

The same procedure used to derive eq 2a-d was invoked to

derive eq 3a-d, which corrects for ²H incorporation of less than

100% in the labeled 4-methylphenols.

k_cat

) X

D0

(3a)

( )

K

k_-1e k₂, i.e., if the substrate were sticky.) For D1, k_2a) k_2b

)

k_cat

X(3[1 - 0.986] + 0.986[2s + p])

(s)k₂and k_2c) (p)k₂, where k_2aand k_2bare rate constants for

)

( )

steps when ¹H is removed, k_2cis the rate constant for ²H

K

D1

3

1

removal, and k₂is the rate constant for removal of H from

X(3[1 - 0.986] + 0.986[2/S + 1/P])

(3b)

unlabeled 4-methylphenol. As a result, the change in magnitude

of the values of steady-state parameters will be due to an

intermolecular isotope effect. The constants s ) 1/S and p )

1/P, where S and P are the intrinsic secondary R-deuterium and

intrinsic primary deuterium isotope effects, respectively. Simi-

larly, for D2, k_2a) k_2b) (sp)k₂(²H removed) and k_2c) (s²)k₂

(¹H removed). Thus, the observed effect is a combination of

inter- and intramolecular isotope effects. The rate constants for

D3 are k_2a) k_2b) k_2c) (s²p)k₂, purely an intermolecular effect.

This analysis also assumes that the rule of geometric mean

3

X([1 - 0.993][2s + p] + 0.993[s²+ sp])

k_cat

)

( )

K

D2

3

X([1 - 0.993][2/S + 1/P] + 0.993[1/S²+ 2/SP])

(3c)

(3d)

3

X([1 - 0.974][s²+ 2sp] + 3(0.974)[s²p])

k_cat

)

( )

K

D3

3

X([1 - 0.974][1/S²+ 2/SP] + 3(0.974)/S²P)

holds: k_HHH/k_DHH) k_HHD/k_DHD) k_HDD/k_DDDand k_HHH/k_HHD

)

3

(17) Northrop, D. B. Anal. Biochem. 1983, 132, 457-461.

(18) Northrop, D. B. In Isotope Effects on Enzyme Catalyzed Reactions;

Cleland, W. W., O’Leary, M. H., Northrop, D. B., Eds.; University Park

Press: Baltimore, MD, 1977; pp 122-152.

(19) Northrop, D. B. In Enzyme Mechanisms from Isotope Effects; Cook,

P. F., Ed.; CRC Press: Boca Raton, FL, 1991; pp 181-202.

a

Table 1. Values of (k_cat/K_S)_Dx

,

^Dx(k_cat/K_S), and Intrinsic Primary (P) and Secondary (S) Deuterium Isotope Effects for 4-Methylphenol and 4-Ethylphenol

(k_cat/K_S)_Dx(µM^-1s^-1

)

b

4-methylphenol

(k_cat/K_S)_D0

(k_cat/K_S)_D1

(k_cat/K_S)_D2

(k_cat/K_S)_D3

^D1(k_cat/K_S)

^D2(k_cat/K_S)

^D3(k_cat/K_S)

S

P

set 1

set 2

set 3

8.06 ( 0.20

8.06 ( 0.17

8.06 ( 0.31

8.06 ( 0.08

5.71 ( 0.14

5.73 ( 0.07

5.45 ( 0.08

5.52 ( 0.12

3.63 ( 0.21

3.75 ( 0.06

3.21 ( 0.03

3.63 ( 0.12

1.14 ( 0.04

1.32 ( 0.01

1.19 ( 0.01

1.16 ( 0.02

1.41 ( 0.05

1.41 ( 0.03

1.48 ( 0.06

1.46 ( 0.03

1.44 ( 0.02

2.22 ( 0.14

2.03 ( 0.06

2.51 ( 0.10

2.22 ( 0.08

2.18 ( 0.04

7.09 ( 0.30

6.11 ( 0.14

6.79 ( 0.27

6.95 ( 0.13

6.63 ( 0.08

0.988 ( 0.029

0.960 ( 0.037

1.039 ( 0.011

1.000 ( 0.049

1.013 ( 0.014

7.52 ( 0.35

6.44 ( 0.25

6.71 ( 0.09

7.27 ( 0.50

6.71 ( 0.08

set 4

weighted av^c

(k_cat/K_S)_Dx(µM^-1s^-1

)

4-ethylphenol

set 1

(k_cat/K_S)_H2

b

(k_cat/K_S)_R-D

(k_cat/K_S)_S-D

47.0 ( 0.8

(k_cat/K_S)_R,S-D

44.1 ( 0.7

(k_cat/K_S)_D2

^R-D(k_cat/K_S)

1.59 ( 0.06

^S-D(k_cat/K_S)

1.49 ( 0.06

^R,S-D(k_cat/K_S)

1.56 ( 0.10

^D2(k_cat/K_S)

S

P

A

70.0 ( 2.4

44.8 ( 2.3

13.7 ( 0.1

13.0 ( 0.2

12.4 ( 0.2

11.5 ( 0.2

5.09 ( 0.18

5.36 ( 0.31

5.62 ( 0.08

6.10 ( 0.11

5.69 ( 0.06

0.927 ( 0.060

0.894 ( 0.078

0.967 ( 0.027

1.030 ( 0.121

1.020 ( 0.055

1.041 ( 0.062

1.158 ( 0.197

1.109 ( 0.216

0.967 ( 0.021

0.996 ( 0.044

5.96 ( 0.16

6.18 ( 0.26

6.03 ( 0.16

5.61 ( 0.71

5.99 ( 0.83

5.86 ( 0.89

5.71 ( 1.11

5.97 ( 1.33

5.98 ( 0.12

6.09 ( 0.23

0.84 ( 0.07

1^d

set 2

70.0 ( 1.1

70.0 ( 0.2

70.0 ( 0.9

46.7 ( 1.0

45.7 ( 0.6

46.4 ( 0.5

40.3 ( 0.3

40.0 ( 0.3

40.6 ( 1.2

1.73 ( 0.03

1.75 ( 0.01

1.72 ( 0.05

1.74 ( 0.01

1.50 ( 0.04

1.55 ( 0.02

1.56 ( 0.03

1.54 ( 0.02

1.70 ( 0.07

1.76 ( 0.01

1.93 ( 0.03

1.77 ( 0.01

0.76 ( 0.03

1^d

set 3

39.8 ( 0.3

36.3 ( 0.3

0.82 ( 0.14

1^d

0.51 ( 0.28

1^d

set 4

weighted av^c

0.78 ( 0.02

1^d

^aThere was an approximately 10% variability in the values of (k_cat/K_S)_Dxbetween sets, although the relative values of these in each set were nearly identical, as reflected in the ratioed values, ^Dx(k_cat/K_S),

in this table. However, this variability does not affect the nonlinear least-squares analysis, since only data with one set are used to calcluate S, P, and A. The variability is due to using different enzyme

preparations over the span of several years. (Also note that k_cat/K_S) k₂/K_D.) ^bTo correct for the variability, the values of (k_cat/K_S)_Dxfor a set were normalized to a constant value of (k_cat/K_S)_D0

.

^cThe weighted

average was calculated from ∑[^D(k_cat/K_S)/σ²]/(∑1/σ²) and the error from [∑1/σ²]^-1/2, where σ are the errors in the table. ^dAssumed values of A.

Isotope Effects and PCMH

J. Am. Chem. Soc., Vol. 121, No. 25, 1999 5869

Scheme 3

The numbers 0.986, 0.993, and 0.974, respectively, are the

fractions of R-d₁in the D1 compound, R-d₂in the D2 compound,

and R-d₃in the D3 compound. Pairs of these k_cat/K_4-MP

equations can be solved for S and P; however, rather than solve

for the parameters in this manner, we determined the values

for S and P by adapting a nonlinear least-squares analysis

program to solve the four equations simultaneously²⁰for each

separate set of k_cat/K_4-MPvalues given in Table 1. This resulted

in four values of S and P, which are also provided in Table 1.

The final values of S and P were determined by weighted

averaging of the four values of each (see footnote to Table 1).

The final values are S ) 1.013 ( 0.014 and P ) 6.71 ( 0.08.

If oxidation of 4-methylphenol proceeds via a hydride

elimination mechanism, then p-quinone methide will form in

the same step in which the R-C-H bond is broken. Also, if the

reaction has a late transition state and if this mechanism is really

operating, then on progressing from the ground state to the

transition state, there might be a change in the force constants

for the C-H bonds at the 2, 3, 5, and 6 positions, and oxidation

of 4-methyl(2,3,5,6-²H₄)phenol would show an isotope effect.

The measured k_cat/K_4-MPisotope effect for 4-methyl(2,3,5,6-

²H₄)phenol was determined to be 1.001 ( 0.022.

Reaction of 4-Ethylphenol with PCMH. Unlike the oxida-

tion of 4-methylphenol by PCMH, the reaction of the enzyme

with 4-ethylphenol is more complex. First, in contrast to an early

observation,¹⁶oxidation of 4-ethylphenol produces not only 1-(4-

hydroxyphenyl)ethanol but also 4-vinylphenol.²¹Additionally,

there are three distinct and potentially stereoselective steps in

the oxidation of 4-ethylphenol to 4-hydroxyacetophenone

(Scheme 3): (1) removal of the pro-R or pro-S hydrogen from

4-ethylphenol; (2) addition of water to the re or si side of the

p-quinone methide; and (3) oxidation of R- or S-1-(4-hydroxy-

phenyl)ethanol. In an early study,¹⁶it was determined that

PCMH oxidation of 4-ethylphenol produces 66% S-1-(4-

hydroxyphenyl)ethanol and 34% of the R-isomer [32% enan-

tiomeric excess (ee) of the S-isomer]. While millimolar amounts

of the alcohol were formed from 3-4 mM 4-ethylphenol, only

micromolar amounts of the alcohol were converted to 4-hy-

droxyacetophenone. Thus, the low enantiomeric excess of the

S-isomer cannot be the result of preferential oxidation of the

S-alcohol to ketone by PCMH. In the previous work, the alcohol

was generated in a reaction mixture that contained PCMH,

4-ethylphenol, PMS (phenazine methosulfate) to reoxidize the

Figure 1. Stereochemical analysis of racemic, diacetylated 1-(4-

hydroxyphenyl)ethanol (top chromatogram) and the diacetylated alcohol

formed on enzymic oxidation of 4-ethylphenol (lower chromatogram).

The samples were chromatographed on a Pirkle type-1-A HPLC

column: n-hexane/2-propanol, 97:3; flow rate, 1 mL/min; 254 nm

detection. The peak at 4.7 min in the lower chromatogram is due to

trace 4-ethylphenol.

enzyme, KCN to minimize enzyme inhibition by PMS, and

catalase. The reaction was carried out at pH 9.5, with shaking

in air for 2 h in order to maximize the concentration of dissolved

O₂, a necessity for oxidation of reduced PMS. Catalase destroys

H₂O₂formed in the oxidation of reduced PMS.¹⁶Unfortunately,

the resulting 1-(4-hydroxyphenyl)ethanol required extensive

purification to remove the other components of the mixture and

base-catalyzed breakdown products of PMS.²¹This and the high

pH of the reaction mixture caused concern about the stereo-

chemical outcome of the experiment; therefore, a new method

was devised to recycle oxidized enzyme at pH 7.6. The new

procedure required far fewer manipulations to obtain pure

alcohol formed by enzymic oxidation of 4-ethylphenol. For this

method, substrate-reduced PCMH was oxidized by horse heart

cytochrome c at low ionic strength, and the resulting reduced

cytochrome was reoxidized by bovine cytochrome c oxidase.

The reaction was stirred in air, and reduced cytochrome oxidase

was oxidized by conversion of O₂to H₂O. After the reaction

was quenched with (NH₄)₂SO₄, protein was removed by

ultrafiltration, and 4-ethylphenol and its reaction products were

isolated by HPLC. In early experiments, the 1-(4-hydroxphenyl)-

ethanol obtained in this manner was acetylated in order to

separate the R- and S-isomers on a Pirkle column (Figure 1).^22,23

Several repeat experiments gave similar results. It was estimated

that the product alcohol was >97% S-isomer (> 94% ee). In

more recent experiments, underivatized alcohol samples were

chromatographed on a Chiracel column^24,25(data not shown;

see Figure 5 for separation of R- and S-isomers on the Chiracel

column), and the results were the same as for the Pirkle column

analyses.

(22) Pirkle, W. H.; Finn, J. M. J. Org. Chem. 1981, 46, 2935-2938.

(23) Kasai, M.; Froussios, C.; Ziffer, H. J. Org. Chem. 1983, 48, 459-

464.

(20) Sherril, D. C.; Tucker, E. E. Am. Lab. 1983, 15, 78-85.

(21) McIntire, W. S.; Bohmont, C. In FlaVins and FlaVoproteins;

Edmondson, D. E., McCormick, D. B., Eds.; Walter de Gruyter: Berlin

and New York, 1987; pp 677-686.

(24) Technical Brochure No. 3 (Chiralcel); Diacel (USA), Inc., Los

Angeles, CA.

(25) Okamoto, Y.; Aburatani, R.; Hatada, K. J. Chromatogr. 1987, 389,

95-102.

5870 J. Am. Chem. Soc., Vol. 121, No. 25, 1999

McIntire et al.

Figure 2. (A) HPLC separation of 4-ethylphenol and its enzymic oxidation products. The reaction of 4-ethylphenol (3 mM) with PCMH was

carried out in 10 mM Tris-HCl, pH 7.6, I ) 0.01, in the presence of the reoxidizing substrate, horse heart cytochrome c. The various phenolic

compounds were isolated as described in the Experimental Procedures section. Shown is a chromatogram of a sample of the reaction mixture that

had incubated at room temperature for 8 h. A Hypersil ODS, 5-µm, 0.46- × 25-cm column was used: 10% (v/v) MeOH/H₂O for 3 min, then from

10 to 40% (v/v) MeOH/H₂O in 12 min; 2 mL/min flow rate; 280 nm detection; 20 µL injected. (B) Time course for oxidation of 4-ethylphenol by

PCMH (see above for conditions). At various times over a 240-min period, samples were removed and processed as described in the Experimental

Procedures section. HPLC analyses of small aliquots for each time-point sample were done as described above. Concentrations were determined

by integrating the peak areas and relating these to the areas of the corresponding peaks of HPLC runs of 20-µL samples containing known concentrations

of 4-ethylphenol, 4-vinylphenol, 1-(4-hydroxyphenyl)ethanol, and 4-hydroxyacetophenone. Plots: 4-ethylphenol, b (left axis); 1-(4-hydroxyphenyl)-

ethanol, 2 (close right axis; label, ALCOHOL); 4-vinylphenol, 9 (close right axis); 4-hydroxyacetophenone, + (far right axis; label, KETONE).

In addition to 1-(4-hydroxyphenyl)ethanol and 4-hydroxac-

etophenone, a third product of enzymic oxidation of 4-ethylphe-

nol was detected by reversed-phase HPLC (Figure 2A).²¹The

third product was shown to be 4-vinylphenol. After purification,

the material was methylated with diazomethane and shown to

have the same UV-visible spectrum and reversed-phase HPLC

properties as commercial 4-vinylanisole.²¹Mass spectral analy-

ses confirmed that 4-vinylphenol had been produced (vide infra).

The time course for formation of the various products from

enzymic oxidation of 4-ethylphenol is shown in Figure 2B. For

the reaction of PCMH with 4-(2-propyl)phenol or 4-(n-propyl)-

phenol, the corresponding R-carbinols and R-alkene formed, but

not the R-carbonyls (it is not possible for 4-(2-propyl)phenol

to form the carbonyl). On enzymic oxidation of 5-indanol and

6-hydroxytetralin, corresponding R-carbinols, R-carbonyls, and

R-alkenes formed.²¹

Isotope Effects and PCMH

J. Am. Chem. Soc., Vol. 121, No. 25, 1999 5871

Two observations are apparent from Figure 2B. First,

4-ethylphenol is not completely consumed in the reaction. Even

after 24 h, the relative amounts of each component are little

changed from those measured at 240 min in Figure 2A. Second,

the ratio of [4-vinylphenol] and [1-(4-hydroxyphenyl)ethanol]

formed on oxidation of 4-ethylphenol is the same at each time

point. To understand the first observation, assays were carried

out with an oxygen electrode. In an assay with cytochrome c

oxidase, 40 µM horse heart cytochrome c, 1.34 mM 4-ethylphe-

nol, and 14 µg/mL of PCMH, the initial rate was 2.5 µmol of

O₂min^-1mg^-1, and the reaction leveled off after about 45 min,

when ∼0.20 µmol of O₂/mL was consumed (at 25 °C,

air-saturated buffer contains 0.26 µmol/mL of O₂²⁶). Inclusion

of 0.1 mM 1-(4-hydroxyphenyl)ethanol or 4-hydroxyacetophe-

none had little effect on the initial rate or the leveling off.

However, inclusion of 0.5 mM 4-vinylphenol reduced the initial

rate dramatically. Preincubation of PCMH with 4-ethylphenol,

1-(4-hydroxyphenyl)ethanol, or 4-hydroxyacetophenone made

no difference. PCMH was not irreversibly inhibited because

addition of saturating [4-methylphenol] to the mixture, after O₂

consumption ceased, gave a return of O₂consumption at a rate

slightly lower than that seen when only 4-methylphenol was

present as substrate. It was concluded that 4-vinylphenol was

acting as a good competitive inhibitor for the 4-ethylphenol

(K_4-EP) 2.6 mM) reaction but a poor inhibitor for the

4-methylphenol (K_4-MP) 16 µM) reaction; i.e., it was assumed

that, for 4-vinylphenol, 16 µM < K_i) K_D< 2.6 mM.

Subsequent steady-state kinetic experiments proved that 4-vi-

nylphenol was a competitive inhibitor for 4-ethylphenol. The

K_ivalue was determined to be 94.3 ( 7.2 µM.

With regard to the second observation from Figure 2B (the

constant ratio [1-(4-hydroxyphenyl)ethanol]/[4-vinylphenol] at

all time points), two explanations are can be advanced. (1) Two

independent, competing pathways lead from 4-ethylphenol to

4-vinylphenol and 1-(4-hydroxyphenyl)ethanol, respectively. In

this case, ratio of the rates (partition ratio) of formation of the

two products will be V_a/V_b) {(k_cat/K)_a[S_a]}/{(k_cat/K)_b/[S_b]}, even

when 4-vinylphenol product inhibition is occurring.²⁷Since S_a

and S_bare both 4-ethylphenol, then V_a/V_b) (k_cat/K)_a/(k_cat/K)_b.

Thus, the partition ratio is independent of [4-ethylphenol]. (2)

4-Vinylphenol and 1-(4-hydroxyphenyl)ethanol are formed from

a common intermediate, i.e., the p-quinone methide in Scheme

3. For this situation, the partition ratio is k_I/k_II(Scheme 3), which

is also independent of [4-ethylphenol].

Figure 3. (A) Time course for the anaerobic reaction of 2.4 mM

4-vinylphenol with reduced PCMH (see the Experimental Procedures

section for details). The plot for the change in [4-vinylphenol] is denoted

by b (left axis), and the plot for the change in [1-(4-hydroxyphenyl)-

ethanol] is indicated by 2 (right axis; label, Alcohol). (B, Inset) Time

courses for the anaerobic reaction of 1-(4-hydroxyphenyl)ethanol with

reduced PCMH. The plot displays the increase in [4-vinylphenol]. The

change in [1-(4-hydroxyphenyl)ethanol] was too small to be measured

reliably.

in the partition ratio. However, this would not be the case if

the two products formed from a common intermediate (step II

in Scheme 3).

To help distinguish between the two mechanisms, PCMH was

anaerobically incubated with 4-vinylphenol or 1-(4-hydroxy-

phenyl)ethanol. Figure 3A shows the results of an experiment

where 2.4 mM 4-vinylphenol was incubated with reduced

PCMH. It is apparent that a very slow conversion of 4-vi-

nylphenol to 1-(4-hydroxyphenyl)ethanol occurred. No such

conversion of 4-vinylphenol resulted when PCMH was absent,

or when oxidized PCMH was present. The second control

reaction was done under aerobic conditions in the presence of

cytochrome c and cytochrome c oxidase so that any reduced

PCMH would be quickly reoxidized.

Figure 3B presents the results of an experiment in which 3.4

mM 1-(4-hydroxyphenyl)ethanol is incubated with reduced

PCMH. In this case, there is a very slow formation of very low

amounts of 4-vinylphenol. 4-Vinylphenol was not formed when

PCMH was excluded. These observations could be rationalized

with the parallel pathway mechanism only if 4-vinylphenol and

1-(4-hydroxyphenyl)ethanol can be converted to 4-ethylphenol

by reduced PCMH. In the reaction mixture containing only 1-(4-

hydroxyphenyl)ethanol at t ) 0, reoxidation of reduced PCMH

by this compound would produce 4-ethylphenol. Since 1-(4-

hydroxyphenyl)ethanol is present in such large excess, all of

the newly oxidized PCMH would be reduced by the alcohol,

forming 4-hydroxyacetophenone. Thus, it is expected that

equimolar amounts of 4-ethylphenol and 4-hydroxyacetophe-

none and very low levels of 4-vinylphenol would be seen.

Additionally, slow reoxidation of reduced enzyme by trace

oxygen did not take place to an appreciable extent, because

Ratios of [1-(4-hydroxyphenyl)ethanol]/[4-vinylphenol] were

determined from HPLC experiments at various times in the

reaction of 4-ethylphenol with PCMH (see Figure 2), and then

these were averaged for each experiment. For 3.23, 1.62, 0.642,

and 0.10 mM initial concentrations of 4-ethylphenol, averaged

ratios, PR1, were 2.11 ( 0.08, 1.96 ( 0.04, 1.96 ( 0.05, and

2.05 ( 0.09, all equivalent within experimental error. These

results support either the parallel[1] pathway or the branched-

[2] pathway mechanism. In a similar manner, the partition ratios

for 4-(1′,1′-²H₂)ethylphenol and 4-(2′,2′,2′-²H₃)ethylphenol were

determined to be 2.53 ( 0.05 (PR2) and 4.05 ( 0.13 (PR3),

respectively. If the parallel pathway mechanism were operating,

and the mechanism in each path were similar, it might be

expected that the ratios for 4-ethylphenol, 4-(1′,1′-²H₂)-

ethylphenol, and 4-(2′,2′,2′-²H₃)ethylphenol would be similar;

i.e., the isotope effects in each path are similar and would cancel

(26) Estabrook, R. W. Methods Enzymol. 1967, 10, 41-47.

(27) Cornish-Bowden, A. Fundamentals of Enzyme Kinetics; Portland

Press: London, 1995; pp 105-110.

5872 J. Am. Chem. Soc., Vol. 121, No. 25, 1999

McIntire et al.

oxidized enzyme formed in this manner would be reduced by

excess 1-(4-hydroxyphenyl)ethanol to form 4-hydroxyacetophe-

none. Almost no 4-hydroxyacetophenone, and no 4-ethylphenol,

was detected in the reaction mixture after 50 h. It is concluded

that 4-vinylphenol and 1-(4-hydroxyphenyl)ethanol interconvert

through the putative p-quinone methide intermediate. This

provides prima facie evidence that these two compounds are

formed by enzymic oxidation of 4-ethylphenol via the branched

pathway mechanism; i.e., 4-ethylphenol is converted to the

p-quinone methide, which can either be hydrated to form 1-(4-

hydroxyphenyl)ethanol or tautomerized to 4-vinylphenol (Scheme

3).

Deuterium Isotope Effects in the Reaction of 4-Ethylphe-

nol with PCMH. Using the mechanism in Scheme 3 and

making some assumptions, it is possible to extract intrinsic

deuterium isotope effects on steps I and II in the scheme. With

4-(1′,1′-²H₂)ethylphenol as substrate, the p-quinone methide

intermediate will be R-deuterated. The rate of conversion of

this intermediate to 4-(1′-²H₁)vinylphenol (k_IIR-d) should be

similar to the that for R-hydrogenated material since the

R-carbon sp²hybridization is maintain in the conversion (i.e.,

k_IIR-d≈ k_IIR-h). On the other hand, conversion of p-quinone

methide to 1-(4-hydroxyphenyl)ethanol involves a change from

sp²to sp³hybridization at the R-carbon and should result in an

inverse R-deuterium isotope effect (i.e., k_IR-d> k_IR-h). The

values for the pertinent partition ratios are PR1 ) k_IR-h/k_IIR-h

) 2.11 ( 0.08 and PR2 ) k_IR-d/k_IIR-d) 2.53 ( 0.05 (see

previous section). Thus, k_IR-h/k_IR-d) ^Dk_IR≈ PR1/PR2 ) 0.83

( 0.04. This is the intrinsic secondary R-deuterium isotope

effect for step I.

When 4-(2′,2′,2′-²H₃)ethylphenol is the substrate, the â-methyl

group of the p-quinone methide is fully deuterated. For step II,

a large primary isotope is expected because a â-H/D is lost. On

the other hand, in step I, the rate would be decreased for the

deuterated intermediate due to a secondary â-deuterium isotope

effect. The secondary effect would result from hyperconjugation

of the deuterons with an R-carbonium center.²⁸It is expected

that the electron-releasing ability of the p-hydroxyl group would

minimize the carbonium ion character of the R-carbon, thus

minimizing the isotope effect. This argument has been used to

explain the â-deuterium isotope effects observed in the sol-

volysis of para-substituted 1-phenylethyl chlorides.²⁹The isotope

effect decreased with the electron-releasing ability of the

aromatic substituent, with 1-(4-methoxyphenyl)(2,2,2-²H₃)ethyl

chloride having the lowest effect (^Dk ) 1.133). The low value

was explained by a decreased “conjugative demand on the â-CH

bond” due to the resonance contribution of structure 1.²⁹

Figure 4. Possible stereo-orientations of 4-ethylphenol at the active

site of PCMH. Hypothetical reactions A and B indicate precise

orientations of the C-H bond being cleaved: Reaction A, pro-S C-H

bond cleaved; reaction B, pro-R C-H bond cleaved. Reaction C depicts

the direction of attack by H₂O/HO^-at the si side of PCMH-bound

p-quinone methide intermediate, to produce the correct product, S-1-

(4-hydroxyphenyl)ethanol. Reaction D is a cartoon of an orientation

where the CH₃group is more or less fixed in the active site; i.e., CH₃).

In this orientation, there is a nearly equal probability that the pro-R or

pro-S C-H bond will be cleaved. X represents the group to which the

hydrogen is transferred, presumably FAD.

the â-deuterium isotope effect for step I in Scheme 3 is ∼1

(i.e., k_Iâ-d≈ k_Iâ-h), then the primary effect for step II is PR3/

PR1 ) (k_Iâ-d/k_IIâ-d)/(k_Iâ-h/k_IIâ-h) ) k_IIâ-h/k_IIâ-d) ^Dk_IIâ) 1.92

( 0.10. In contrast, if we assume a value of 1.3 as a maximum

of the secondary â-effect (the maximum measured for solvolysis

of substituted 1-phenylethyl chlorides),²⁹then ^Dk_IIâ≈ 1.92/(1.3)

≈ 1.5.

In a prior study, the intrinsic isotope effects for the oxidation

of 4-(1′,1′-²H₂)ethylphenol by PCMH were found to be 5.21 (

0.20 [)^D(k_cat/K_m), steady-state kinetic parameters] and 4.94 (

0.19 ()^Dk from stopped-flow kinetic studies).¹⁴However, as

with 4-methylphenol, this effect results from participation of

the intrinsic primary and intrinsic secondary R-deuterium effects.

To isolate these two effects, the following compounds were used

as substrates in steady-state kinetic experiments: 4-ethylphenol,

R-(+)-4-(1′-²H₁)ethylphenol, S-(-)-4-(1′-²H₁)ethylphenol, R,S-

(()-4-(1′-²H₁)ethylphenol, and 4-(1′,1′-²H₂)ethylphenol. The

kinetic measurements were done by varying [4-ethylphenol] and

using 0.592 mM PES in 50 mM Tris-HCl, pH 7.6, I ) 0.05 at

25 °C. Steady-state kinetic parameters were measured in four

independent experiments. To minimize systematic errors, dif-

ferent substrate and PCMH solutions were used in each set of

experiments. Since the 4-ethylphenol/PES reaction obeyed ping-

pong-type kinetic behavior, accurate measurements of k_cat/K_4-EP

D

at a single [PES] were obtained, and ^D(k_cat/K_4-EP) ) k_intrinsic

because the commitment to catalysis factor, C, is zero.¹⁴The

isotope effects for the four experiments are provided in Table

1. Surprisingly, all the R-monodeuterated ethylphenols, regard-

less of stereochemical nature, gave nearly identical isotope

effects. This can only be rationalized by assuming that H/D in

the R- and S-positions have an almost equal chance of being

removed; that is, the reaction is not very stereoselective. If the

H/D being removed at the R-position must be precisely

positioned, then two binding orientations for 4-ethylphenol can

be envisioned as depicted in A and B in Figure 4. Both

orientations would have a nearly equal probability of existing.

The resulting p-quinone methides for each would have opposite

orientations; however, this conflicts with the fact that >97%

(Compare structure 1 with the p-quinone methide structure in

Scheme 3.) Since the conjugative effect of a p-hydroxyl group

is greater than that of a p-methoxy group, the â-deuterium

isotope effect for the p-quinone methide intermediate in the

PCMH reaction could be lower than 1.13. If we assume that

(28) Melander, L.; Saunders: W. H. Reactions Rates of Isotopic

Molecules; John Wiley & Sons: New York, 1980.

(29) Shiner, V. L. In Isotope Effects in Chemical Reactions; Collins, C.

J., Bowman, N. S., Eds.; Van Nostrand Reinhold: New York, 1970; Chapter

2, pp 90-159.

Isotope Effects and PCMH

J. Am. Chem. Soc., Vol. 121, No. 25, 1999 5873

Scheme 4

k_cat

) X(1 + A)

H2

(5a)

( )

K

k_cat

1

S

1

P

1

S

) X 1 + 0.967 - 1 + 0.83

-

+ A 1 +

( )

(

[

(

)

]

[

K

R-D

1

P

1

P

0.967 - 1 + 0.83

-

(5b)

)

] ]

(

[

S

k_cat

1

S

1

of S-1-(4-hydroxyphenyl)ethanol is formed; i.e., H₂O/HO^-

always attacks at the si face of the R-carbon of the p-quinone

methide in the active site of PCMH (reaction C, Figure 4). It is

proposed that the CH₃group is fixed, and the C-H_Rand C-H_S

bonds are cleaved with nearly equal propensity, resulting in

transfer of either H to the same group in PCMH (reaction D,

Figure 4).

) X 1 + 0.983 - 1 + A 1 + 0.983 - 1

( )

(

)

[

]

[

(

)

]

K

S-D

P

(5c)

(5d)

k_cat

X(1 + A)(0.971[1/S + 1/P - 2] + 2)

)

( )

K

R,S-D

2

k_cat

)

( )

Scheme 4 presents the mechanism that was invoked to derive

equations for k_cat/K for 4-(1′,1′-²H₂)ethylphenol, R-4-(1′-²H₁)-

ethylphenol, S-4-(1′-²H₁)ethylphenol, and R,S-4-(1′-²H₁)ethyl-

phenol. The scheme presents two paths, one proceeding with

removal of the pro-R hydrogen (ES_R) and the other with removal

of the pro-S hydrogen (ES_S). It is assumed there is no isotope

effect on binding. The most general situation presumes dif-

ferential binding for 4-ethylphenol when either the R or S C-H

bond is cleaved. The difference in binding and dissociation for

the two branches is provided by δ and γ. It is also assumed

that the rates of cleavage of the two C-H bonds are different

(k₂vs ꢀk₂). E′P represents an equivalent (or nearly equivalent)

enzyme/p-quinone methide species for each path. From the

scheme, the following expressions were derived for k_cat/K for

4-ethylphenol (H2), 4-(1′,1′-²H₂)ethylphenol (D2), R-4-(1′-²H₁)-

ethylphenol (R), S-4-(1′-²H₁)ethylphenol (S), and R,S-4-(1′-²H₁)-

ethylphenol (RS), and assuming C ) k₂/k_-1) 0:¹⁴

K

D2

X(1 + A)([1 - 0.965][1/S + 1/P] + 2(0.965)(1/SP))

(5e)

2

(See the Experimental Procedures section for the method used

to derive these equations.) In these equations, 0.967, 0.983,

0.971, and 0.965, respectively, are the fractions of the d₁

derivative in R-D, S-D, and R,S-D and the fraction of the d₂

derivative in D2. In eq 5b, 0.83 is the fraction of the R-D in the

“R-isomer”. These five k_cat/K values were measured in four

separate experiments (Table 1). For each set, the values of S,

P, and A were determined using the same nonlinear regression

analysis used for the 4-methylphenol case. The final values of

these parameters are the weighted averages of the four values

given in Table 1. These average values are S ) 0.967 ( 0.021,

P ) 5.98 ( 0.12, and A ) 0.78 ( 0.02. The analysis was redone

assuming A ) 1. In this case, the weighted average values of S

and P are 0.996 ( 0.044 and 6.09 ( 0.23, respectively.

In the above analysis, it was assumed that the values of S

were identical and the values of P were identical for both

branches in Scheme 4. Alternatively, it can be assumed that

the values of S are different and the values of P are different.

Nonlinear least-squares analyses with the appropriate equations

failed to converge to a minimum sum-of-squared residuals,

regardless of the initial estimates of the parameters.

Oxidation of 1-(4-Hydroxyphenyl)ethanol by PCMH. As

previously reported, PCMH can oxidize 1-(4-hydroxyphenyl)-

ethanol to 4-hydroxyacetophenone.^13,15,21Incubation of PCMH

with racemic 1-(4-hydroxyphenyl)ethanol, and analysis of the

chemical (reversed-phase HPLC) and stereochemical (HPLC

analysis of the remaining alcohol with a Pirkle column) nature

of the reaction mixture as a function of time, indicated that the

S-isomer was preferentially oxidized to the R-isomer.²¹More

recently, this reaction was monitored using the Chiracel OB

column rather than the Pirkle column. The results of the analysis

are shown in Figure 5. Figure 5A shows the separation of the

R- and S-isomers that is achieved on the Chiracel column. Figure

5B present the progress curves for the reaction. Notice that the

total [1-(4-hydroxyphenyl)ethanol] and [R-1-(4-hydroxyphenyl)-

ethanol] converge after prolonged reaction time. Also shown

k_cat

X(1 + (ꢀ/γ)C + A(1 + C))

(1 + (ꢀ/γ)C)(1 + C)

)

H2

) X(1 + A) (4a)

( )

K

k_cat

X(p + (ꢀ/γ)spC + A(s + spC))

(1 + (ꢀ/γ)sC)(1 + pC)

)

) X(p + sA)

(4b)

( )

K

R-D

k_cat

X(p + (ꢀ/γ)spC + A(p + spC))

(1 + (ꢀ/γ)pC)(1 + sC)

)

) X(s + pA)

(4c)

( )

K

S-D

k_cat

X(1 + A)(p + s)

) {COMPLEX} )

(4d)

( )

K

R,S-D

2

k_cat

spX(1 + (ꢀ/γ)spC + A(1 + spC))

(1 + (ꢀ/γ)spC)(1 + spC)

)

) spX(1 + A)

( )

K

D2

(4e)

In these equations, the parameter A ()δꢀ/γ) is a partition ratio,

which is equal to (k₂/K_D)_S/(k₂/K_D)_R) (k_cat/K)_S/(k_cat/K)_R, since

k₂/K_D) k_cat/K for 4-ethylphenol, and p ) 1/P and s ) 1/S,

where P and S are the intrinsic primary deuterium isotope effect

and the secondary R-deuterium isotope effect, respectively.

Further, it is assumed that the ground states and transition states

for the two pathways are essentially the same; thus, for both

paths, values of S are the same, and values of P are the same.

These equations can be modified to incorporate corrections for

less than 100% deuterium content and stereochemical purity

(“S-isomer” is assumed to 100% S, and “R-isomer” is assumed

to be 83% R, based on the optical rotation experiments; see the

Experimental Procedures section):

in Figure 5B is a plot of ee (%) as a function of time. The data

in this figure were analyzed using the equation T ) Rⁿ/R₀

+

n-1

R (see the Experimental Procedures section for derivation),

where T is the concentration of the total remaining alcohol, R

is the concentration of the remaining R-isomer, R₀is the initial

R-isomer concentration, and n ) (k_cat/K_m)_S/(k_cat/K_m)_R, the ratio

of k_cat/K_mfor the S- and R-isomers. The parameter n can be

thought of as a partition or selectivity ratio for the isomers. This

5874 J. Am. Chem. Soc., Vol. 121, No. 25, 1999

McIntire et al.

faster than the R-isomer. Data obtained using the Pirkle column

provided values of n ) 1.57 ( 0.02 and 1.45 ( 0.03 for the

oxidation of initially racemic 1-(4-hydroxyphenyl)ethanol and

1.39 ( 0.04 for oxidation of initially racemic 1-(4-hydroxyphe-

nyl)(1-²H₁)ethanol. Since binding of these alcohols to PCMH

was very slow and rate limiting, it was not possible to determine

the isotope effects from steady-state data.

Discussion

Herein we describe a new, quick, and convenient method for

extracting intrinsic primary and intrinsic R-secondary deuterium

isotope effects from k_cat/K values determined from steady-state

kinetic measurements using unlabeled and mono-, di-, and

trideuteriomethyl analogues of 4-methylphenol, or using unla-

beled and mono- and dideuteriomethylene derivatives of 4-eth-

ylphenol. These analyses can only be done if the commitment

to catalysis factor, C ()k₂/k_-1) is very small compared to the

intrinsic isotope effects. To determine the effect a finite

commitment to catalysis would have on the values of P and S,

calculations were carried out with assumed, small values of C.

The equations in equation set 4 (4-ethylphenol) were written to

include a constant value of C from 0 to 0.4. For each value of

C, nonlinear least-squares analysis of the equations in this set

was carried out as if the assumption C ) 0 were valid. This

analysis provided new values of S, P, and A. The results showed

that apparent values of P and S increased by about 3-4% as C

increased from 0 to 0.2 (data not shown). Earlier, it was reported

D

that the (k_cat/K) value for 4-(²H₃)methylphenol and the value

^Dk₂(the intrinsic effect) determined by stopped-flow measure-

ments were identical.¹⁴As a result, we believe C is very small,

and likely smaller than 0.2. However, even a value of C as high

as 0.2 would not significantly alter conclusions drawn from an

interpretation of the values of S and P. A similar analysis was

done for 4-methylphenol isotope effects, and the conclusion

concerning the impact of C is the same.

4-Methylphenol Oxidation by PCMH. Analysis of the

steady-state kinetic data provided values of the intrinsic primary

(P) and intrinsic secondary (S) R-deuterium isotope effects for

4-methylphenol: P ) 6.71 ( 0.08 and S ) 1.013 ( 0.014.

Ours is not the first report of the concurrent determination of

primary and R-secondary deuterium isotope effects. Intra-/

intermolecular isotope effect studies of the oxidation of methyl

groups of toluene^5,6and n-octane by cytochrome P-450,⁷and

oxidation of the methyl group of 4-methylanisole by chloro-

peroxidase,⁸have also provided primary and R-secondary kinetic

isotope effects. For these studies, products were isolated,

derivatized, and analyzed by mass spectrometry. In contrast,

the method reported herein involved direct spectrophotometric

steady-state assays. The procedure requires neither extraction

nor derivatization and makes use of a good recording UV-

visible spectrophotometer of the type available in biochemistry

laboratories, and individual assays can be done in a few minutes.

To our knowledge, ours is the first study to concurrently

determine S and P via a direct steady-state kinetic method

involving inter-/intramolecularly labeled substrates.

Figure 5. (A) HPLC separation of the remaining R- and S-1-(4-

hydroxyphenyl)ethanol during a reaction started by mixing racemic

alcohol and PCMH. Chromatography of each 5-µL sample was

accomplished on a Chiracel OB column with 1 mL/min flow rate, using

92:8 (v/v) n-hexane/2-propanol, and 280 nm detection. Shown are

chromatograms of the alcohol extracted from the reaction mixture at t

) 0, 5, and 22 h (top to bottom), respectively. The increases in

magnitude of artifact peaks at retention times 3.5 and 12.8 min are a

result of increased detector sensitivity, which was required since

progressively less material was extracted as the reaction proceeded.

Graph B presents the time course for the reaction. The solid lines

represent the remaining concentrations of R- and S-1-(4-hydroxyphe-

nyl)ethanol (b) and of R-1-(4-hydroxyphenyl)ethanol (+), and the

dashed line represents the remaining % enantiomeric excess (ee) of

the R-isomer (2).

The value of P for 4-methylphenol is in the range expected

for a semiclassical primary isotope effect, which suggests, but

does not prove, that tunneling is absent during H/D trans-

fer.^2,28,30,31Significant tunneling could inflate the value of

equation was fit by nonlinear regression with T as the dependent

variable, R as the independent variable, and R₀as constant. The

analysis provided n ) 1.50 ( 0.01. This value indicates that,

when the R-isomer and S-isomer are presented to PCMH in

equimolar concentrations, the S-isomer is oxidized 1.5 times

(30) Kresge, A. J. In Isotope Effects on Enzyme Catalyzed Reactions;

Cleland, W. W., O’Leary, M. H., Northrop, D. B., Eds.; University Park

Press: Baltimore, MD, 1977; pp 37-63.

(31) Su¨hnel, J.; Schowen, R. I. In Enzyme Mechanisms from Isotope

Effects; Cook, P. F., Ed.; CRC Press: Boca Raton, FL, 1991; pp 3-35.

Isotope Effects and PCMH

J. Am. Chem. Soc., Vol. 121, No. 25, 1999 5875

the primary effect out of the semiclassical range: ^Dk > 7-10.

In the absence of tunneling, the large primary effect also

suggests a symmetrical transition state.³¹An R-secondary effect

of ∼1 implies that significant tunneling, accompanied by

coupled motion for the leaving and remaining H/D, does not

take place. Significant coupled motion plus tunneling would

increase the secondary effect and would decrease the primary

effect.²Interestingly, the calculated semiclassical primary isotope

effect for the reaction C-CH₂CH₂Cl + HO^-f CCHdCH₂+

Cl^-+ H₂O increased from 5.03 to 7.07 when tunneling was

included for a symmetrical transition state,³²whereas the

R-secondary effect changed little (from 1.048 to 1.053). As

progressively more coupled motion was added to the model in

the absence of tunneling, the semiclassical primary effect

decreased significantly. The corresponding effect with tunneling

varied somewhat but tended to increase. On the other hand, the

semiclassical R-secondary effect did not change much as

coupled motion increased in the absence of tunneling, but it

increased significantly for the tunneling model as coupling

increased (from 1.03 to 1.27).³²For PCMH, several scenarios

present themselves: (1) a symmetrical transition state with

modest tunneling but no coupled motion; (2) a transition state

with coupled motion without tunneling; and (3) a mechanism

with an early transition state without coupled motion but with

significant tunneling.

for a late transition state).^29,33Further, secondary R-deuterium

kinetic isotope effects of 1.13-1.30 were reported for solvolysis

of meta- and para-substituted 1-phenylethyl chlorides. The lack

of significant variability of the effect with substitution suggested

that the “tightness of binding of the R-deuterium in the transition

state is independent of the electrophilicity of the resulting

carbonium ion”. Further, “...the R-deuterium effect is nearly

constant even though the amount of transition state double bond

character must increase appreciably between 1-phenylethyl

chloride and 1-(4-methoxyphenyl)ethyl chloride” ²⁹(see struc-

ture 1). If the proposed mechanism is operating for PCMH, a

small secondary R-deuterium effect could indicate an early

transition state; however, the magnitude of the primary effect

suggests a rather symmetrical transition state, although, as

mentioned earlier, an early transition state with tunneling could

result in a large primary and small secondary effect.

One way to detect H/D tunneling is to measure S and P at

various temperatures. The Arrhenius equations for the H- and

D-labeled substrates are ln(k_H) ) ln(A_H) + E_aH/(RT) and ln(k_D)

) ln(A_D) + E_aD/(RT), where A is the Arrhenius prefactor and

E_ais the activation energy. Without tunneling, A_H) A_D, and

plots of ln(k_H) vs 1/T and ln(k_D) vs 1/T should intersect the

ordinate at the same point.²Unfortunately, the useful temper-

ature range for PCMH is very narrow. While ^D3(k_cat/K_4-MP) (7.03

( 0.41) is apparently equal to the intrinsic effect (^D3k₂at 25 °C

and pH 7.6), at 6 °C, ^D3(k_cat/K_4-MP) ) 3.43 ( 0.15 is

considerably lower than the measured intrinsic effect (7.05 (

0.22) at this temperature.¹⁴At 6 °C, C ) k₂/k_-1) 1.49 is

considerably greater than 1. Additionally, PCMH is unstable

above 35 °C. Therefore, the feasible temperature range for k_cat/

K_4-MPmeasurements would be 20-35 °C, a range that is too

narrow for long extrapolation to 1/T ) 0 for ln(k) vs 1/T plots.

Another way to determine if tunneling is present requires

measurement of (k_cat/K_4-MP)_H/(k_cat/K_4-MP)_Tand (k_cat/K_4-MP)_D/

(k_cat/K_4-MP)_T(subscript T represents tritium).²Obviously, it is

not practical (or very safe) to carry out spectrophotometric

steady-state assays with tritium-labeled substrates.

The original hypothetical mechanism for PCMH required

heterolytic cleavage of a C-H bond of the methyl group, with

H^-as the leaving group. This mechanism was formulated

because the p-hydroxy group would facilitate H^-expulsion,

particularly if 4-methylphenol were bound in the phenolate form.

The negative charge could be destabilized by other negative

charges of PCMH in the vicinity of phenolate oxygen, or by

the charged oxygen localized in a hydrophobic environment.

Either or both of these conditions would force electron density

on the phenolate oxygen into the benzene ring of 4-methylphe-

nol, with concomitant weakening of the methyl C-H bond, thus

facilitating H^-elimination (structure 2). This would result in

formation of the p-quinone methide (see Scheme 1). In this

mechanism, there would be a change from sp³to sp²hybridiza-

tion at the benzylic carbon. Equilibrium isotope effects for

analogous E2 eliminations indicate that a maximal secondary

R-deuterium isotope effect of 1.12-1.20 is expected (values

A mechanism involving proton transfer followed by electron

transfer is ruled out because of the high pK_aof the R-C-H.

The pK_aof the methyl group of toluene is 28-35³⁴or 54 (in

acetonitrile), and the pK_aof p-methylanisole is 55 (in acetoni-

trile).³⁵The resonance effect of the p-OH/O^-would raise the

pK_a.

Alternative mechanisms involve substrate-based radical for-

mation. In one version, H^•is generated by homolytic cleavage

of an R-C-H bond of the methyl group, leaving an sp³carbon-

centered radical. (While carbon-centered radicals can have either

sp³or sp²hybridization,³⁶for the substrates used in this study,

this is not very feasible, unless the enzyme environs at the active

site enforce an sp³structure.) Little change in hybridization

would result in a low intrinsic R-secondary deuterium isotope

effect.

Another variation of a radical mechanism assumes rapid

electron abstraction from -OH or -O^-of 4-methylphenol to

leave a “stable” phenoxy radical. This is followed by slow

explusion of H^•from the methyl group (Scheme 5). This radical

mechanism is untenable if the observation is valid that electron

transfer from FAD to heme in PCMH is much faster than any

bond-breaking steps. The first electron removed (step 1, Scheme

5) will convert FAD to semiquinone radical, but, presumably,

the electron will very rapidly transfer to heme. As a result, the

rate of heme reduction will not be coupled to R-C-H/D bond

breakage and will not be sensitive to isotopic substitution. This

is contrary to the observation that the rate of heme reduction in

PCMH, apparently, fully reflects the isotope effect.¹⁴Thus, if

this radical mechanism is operating, homolytic cleavage of the

R-C-H/D bond must occur before electron transfer from FAD^•-

to heme and be rate limiting.

The driving force for removal of a single electron from

substrate by FAD is not apparent. Electron transfer from FAD^•-

(33) Anderson, V. E. In Enzyme Mechanisms from Isotope Effects; Cook,

P. F., Ed.; CRC Press: Boca Raton, FL, 1991; pp 389-417.

(34) Reutov, O. A.; Beletskaya, I. P.; Butin, K. P. CH-Acids; Pergamon

Press: New York, 1978.

(35) Sim, B. A.; Griller, D.; Wayner, D. D. M. J. Am. Chem. Soc. 1989,

111, 754-755.

(36) Nonhebel, D. C.; Tedder, J. M.; Walton, J. C. Radicals; Cambridge

University Press: Cambridge, England, 1979.

(32) Saunders, W. H. J. Am. Chem. Soc. 1985, 107, 164-169.

5876 J. Am. Chem. Soc., Vol. 121, No. 25, 1999

McIntire et al.

Scheme 5

to heme in the absence of substrate is quite fast, at 200 s^{-1 37}

However, the electron tunnels through 13 σ bonds and “jumps”

2.96 Å in its journey from flavin to the heme iron of the

cytochrome subunit of PCMH.³⁸As a result, there must exist a

large thermodynamic driving force for electron transfer from

flavin to heme. Therefore, the one-electron E₇° for FAD bound

to PCMH is expected to be much lower than +248 mV, the

potential for the heme.³⁹On the other hand, the one-electron

reduction potential at pH 7.0 for the 4-methylphenol radical is

+870 mV.⁴⁰Thus, it seems unlikely that FAD could extract a

single electron from the substrate. In addition, as discussed

elsewhere,⁴¹protein-bound ground-state flavin does not have

the reactivity for H^•abstraction. Such an abstraction could be

accomplished by another reactive group, X, on the enzyme,

which has a homolytic X-H bond dissociation energy greater

than that for 4-methylphenol (86 kcal/mol, gas phase).⁴⁰This

group could be an amino acid-based radical. However, there is

absolutely no evidence for this type radical in PCMH (e.g.,

electron spin resonance measurements). Finally, the 4-meth-

ylphenol dissociation energy quoted above is for the O-H bond

of 4-methylphenol. It is expected that the R-C-H homolytic

bond dissociation energy will be larger.

.

Last, mechanisms involving a covalent FAD/substrate were

considered. For one such mechanism (Figure 6A), rather than

direct formation of p-quinone methide, the negative charge on

the para oxygen of the substrate is forced into to the benzene

ring, which enforces a direct and rapid nucleophilic attack on

FAD by the ring carbon at the 4-position of 4-methylphenol.

The attack can be at the 4a-position of the isoalloxazine ring

(see Figure 6) or the N5-position, resulting in a cyclohexadi-

enone-FAD intermediate. This step is followed by slow, base-

catalyzed abstraction of substrate R-proton, with concerted

scission of the cyclohexadienone-FAD bond. This reaction

yields the p-quinone methide intermediate and two-electron-

reduced FAD. Note that, in this mechanism, the rate-determining

heterolytic R-C-H/D bond cleavage would be reflected in the

rapid reduction of heme by two-electron-reduced FAD. A

variation of this mechanism involves rapid attack of FAD by

the phenolate oxygen (Figure 6B), followed by slow R-proton

abstraction, concerted oxygen-FAD bond cleavage, which

results in free p-quinone methide.

Figure 6. Possible mechanisms for oxidation of 4-methylphenol

involving covalent flavin-substrate intermediates.

secondary R-deuterium isotope effect was 0.967 ( 0.021, or

0.996 ( 0.044 when A was set equal to 1. Because the primary

and secondary effects are similar to those found for the

4-methylphenol/PCMH reaction, mechanistic arguments similar

to those presented for 4-methylphenol can be advanced.

In our analysis of the steady-state kinetic results, it was

assumed that values of P for both branches of the reaction in

Scheme 4 were the same, and this was also true for the values

of S for both branches. Additionally, it was assumed that each

branch has different rate constants for substrate binding and

dissociation and catalysis. With these assumptions, it was

possible to extract the P and S from the data. It was impossible

to determine values of P and S, assuming these values are

different for the two paths of Scheme 4, regardless of any

assumption concerning the relative rates of substrate binding

and dissociation, and catalysis. It is possible that the true intrinsic

primary and secondary effects are somewhat different for each

path.

4-Ethylphenol Oxidation by PCMH. For this reaction, a

large intrinsic primary deuterium isotope effect was found (5.98

( 0.12, or 6.09 ( 0.23 when A was set equal to 1), again

suggesting a rather symmetrical transition state. The intrinsic

(37) Bhattacharyya, A.; Tollin, G.; McIntire, W. S.; Singer, T. P.

Biochem. J. 1985, 228, 337-345.

It is interesting that the stereochemistry of the products of

each branch are identical because ∼100% of (-)-S-1-(4-

hydroxyphenyl)ethanol is formed; i.e., the p-quinone methide

is always attacked by H₂O/HO^-on the si side of the R-carbon.

This suggests that the transition states for both paths of Scheme

4 are the same or nearly so and suggests that the binding of

4-ethylphenol is essentially the same for both paths (see Figure

(38) Kim, J.; Fuller, J. H.; Kuusk, V.; Cunan, L.; Chen, Z.-w.; Mathews,

F. S.; McIntire, W. S. J. Biol. Chem. 1995, 270, 31202-31209.

(39) Hopper, D. J. FEBS Lett. 1983, 161, 100-102.

(40) Lind, J.; Shen, X.; Eriksen, T. E.; Mere´nyi, G. J. Am. Chem. Soc.

1990, 112, 479-482.

(41) Walker, M. C.; Edmondon, D. E. Biochemistry 1994, 33, 7088-

7098.

Isotope Effects and PCMH

J. Am. Chem. Soc., Vol. 121, No. 25, 1999 5877

using a 5-µm Ultrasphere-ODS (octadecylsilyl-derivatized silica gel)

0.46- × 25-cm column (column 1) (Beckman Instruments, Inc.,

Fullerton, CA), a Spherex 3-µm, C-18, 0.46- × 7.5-cm column (column

2), or a Hypersil 3-µm, ODS, 0.46- × 7.5-cm column (column 3)

(Phenomenex, Torrance, CA). A Beckman Instruments, Inc. model 332

gradient liquid chromatography system and a Kratos Analytical

Instrument Spectroflow 757 detector (Ramsey, NJ) were used. Unless

otherwise stated, the flow rate was 1 mL/min, and effluents were

monitored at 254 or 280 nm; and (C) mass spectral analyses done at

the Mass Spectrometry Facility at the University of California, San

Francisco. All reported melting points and boiling points are uncor-

rected.

4, reaction D). If this is true, then there is a nearly equal

probability for removal of the pro-R or pro-S hydrogen of

4-ethylphenol (A, the flux ratio for the S-isomer/R-isomer paths

) 0.78).

It may be more accurate to think of the PCMH chemical

mechanism in terms of a nonspecific R-hydrogen “explusion”

from the substrate, rather than a specific hydrogen “abstraction”.

If the phenolate forms of 4-methylphenol and 4-ethylphenol bind

to PCMH, then the negative charge on the substrate oxygen

would be destabilized if its immediate environment is hydro-

phobic and/or there is a strong unfavorable electrostatic inter-

action. The unfavorable interaction would be minimized by

forcing the negative charge into the benzene ring of substrate,

thereby increasing its quinonoid character and necessarily

weakening an R-C-H bond. Eventually, there is an indiscrimi-

nant explusion of H^-, and the closest electrophilic center [e.g.,

the N5-position of enzyme-bound FAD] would be the recipient

of H^-. In fact, as the C-H bond weakens, the reaction will be

facilitated by increasing interaction between the increasing δ-

on the hydrogen and the δ+ of an electrophilic center of the

flavin.

PCMH optimally oxidizes 4-methylphenol; therefore, it can

be envisioned that binding of 4-ethylphenol in the active site

of the enzyme involves less than optimal steric interactions. This

is reflected in K_Dvalues for 4-methylphenol (16 ( 3 µM) and

for 4-ethylphenol (2.17 ( 0.38 mM). Possible “steric strain”

could be relieved in the 4-ethylphenol transition state, and the

relief could be greater in reactions with R-secondary C-D. This

greater relief is due to the shorter effective length of the C-D

bond relative to the C-H bond and leads to lowering of S.⁴²

Similarly, it can be argued that steric interactions allow

deuterated 4-ethylphenol slightly closer approach to FAD and/

or other catalytic groups than the nondeuterated form in a highly

structured, crowded active site. This would result in slightly

more favorable interaction(s) in the isotopically sensitive step.

If this is true, then the underlying assumption that there are no

isotope effects for binding may be incorrect. The magnitudes

of errors for K_dvalues measured by stopped-flow for 4-eth-

ylphenol¹⁴precludes the determination of small isotope effects

for binding.

Purifications. The A form of PCMH was isolated from P. putida,

N.C.I.M.B. 9869 by a published method.⁴³Cytochrome c oxidase was

purified from beef heart mitochondria to the red/green split stage.⁴⁴

4-Ethylphenol was purified as described earlier.¹⁴It was pure as

judged by NMR and HPLC, t_R) 7.22 min (100%, column 1, H₂O/

CH₃CN, 1:1, v/v) and 1.84 min (100%, column 3, H₂O/CH₃CN, 3:2,

v/v). Mass spectrum: m/z (relative intensities) 122 (M⁺, 40) 107 (100),

77 (20).

4-Methylphenol and 4-methyl(2,3,5,6-²H₄)phenol were purified by

subliming twice under 0.1 mmHg at 20-32 °C. Mass spectral analysis

of deuterium content for 4-methyl(2,3,5,6-²H₄)phenol: 94.7% ²H₄, 4.8%

²H₃, 0.4% ²H₂, and 0.2% ²H₁, which translated to a total of 98.5 atom

% ²H. Both samples were deemed pure by HPLC, R ) 5.2 min (100%,

column 1, H₂O/CH₃CN, 1:1, v/v) and 1.33 min for 4-methylphenol

(100%, column 3, H₂O/CH₃CN, 3:2, v/v). For 4-methylphenol:¹⁴NMR

2

(C HCl₃) δ 2.2 (s, 2.9, CH₃), 6.16 (s, 1.0, OH), 6.7 and 7.0 (2d, 4.0,

Ar-H); mass spectrum, m/z (relative intensities) 108 (M⁺, 96), 107

(100), 91 (7.1), 90 (8.7), 79 (15.7), 78 (6.3), 77 (27.4).

Purifications of other substrates are described with the syntheses

presented in the following sections. Syntheses of 4-(²H₃)methylanisole,

4-(²H₂)methylanisole, and 4-(²H₁)methylanisole, similar to those used

in the syntheses of 4-(²H₃)methylphenol, 4-(²H₂)methylphenol, and

4-(²H₁)methylphenol,¹⁴are also described elsewhere.⁸

4-(²H₃)Methylphenol and 4-(1′,1′-²H₂)ethylphenol. The syntheses

are described elsewhere.¹⁴The samples were purified by twice

subliming. The samples were pure by HPLC analyses: 4-(²H₃)-

methylphenol, R ) 5.33 min (100%, column 1, H₂O/CH₃CN, 1:1, v/v)

and t_R) 1.29 min (100%, column 3, H₂O/CH₃CN, 3:2, v/v); 4-(1,1-

²H₂)ethylphenol, R ) 7.19 min (100%, column 1, H₂O/CH₃CN, 1:1),

t_R) 4.82 min (100%, column 2, H₂O/CH₃CN, 7:3, v/v) and 1.88 min

(100%, column 3, H₂O/CH₃CN, 3:2, v/v). NMR: 4-(²H₃)methylphenol

2

(C²HCl₃), δ 6.27 (s, 1.1, OH), 7.08 (2d, 4.0, Ar-H), 2.23 (s, H₂CH),

estimated methyl hydrogen content, 0.6%; 4-(1′,1′-²H₂)ethylphenol (C²-

HCl₃), δ 1.20 (s, 2.9, CH₃), 6.45 (s, 1.0, OH), 7.13 (dd, 4.0 Ar-H), ∼2

(multiplet, -²HCH-), estimated methylene hydrogen content, 1-2%.

Mass spectra: m/z (relative intensities), 4-(²H₃)methylphenol, 111 (M⁺,

100), 110 (57), 109 (47.5), 94 (3.8), 93 (6.2), 92 (4.9), 83 (5.9), 82

(12.4), 81 (6.6), 80 (5.9); 4-(1′,1′-²H₂)ethylphenol, 124 (M⁺, 40), 109

(100), 79 (10), 78 (10). Mass spectral analysis of deuterium content:

Experimental Procedures

Materials. 4-Methylphenol (99+%, gold label), 4-ethylphenol

(97%), 4-hydroxyactophenone (99%), 4-methoxybenzyl alcohol (98%),

4-vinylanisole (97%), deuterium chloride (37% solution in D₂O, 99

atom % D, gold label), and LiAlH₄were from Aldrich Chemical Co.

(St. Louis, MO). Other materials and sources were as follow: 4-anisate

2

1

4-(²H₃)methylphenol, 97.4% H₃, 2.3% H₂, 0.1% H, and 0.2% H₃,

2

methyl ester, Eastman Kodak Co. (Rochester, NY); H₂O (99.8 atom

for a total of 98.9 atom % H; 4-(1′,1′-²H₂)ethylphenol, 96.5% H₂,

2

% H), Stohler/KOR Stable Isotopes (Cambridge, MA); LiAl²H₄(99

2

1

atom % ²H) , KOR Isotopes (Cambridge, MA); di(²H₃)methyl sulfoxide

(99.5 atom % ²H), Diaprep, Inc. (Atlanta, GA); 4-methyl(2,3,5,6-²H₄)-

3.3% H₁, and 0.2% H₃, which translated to a total of 98.2 atom %

²H.

2

phenol (98.6 atom % H), tetramethylsilane, and C²HCl₃(99.8 atom

4-(²H₂)Methylphenol. 4-Methoxy(1′,1′-²H₂)benzyl chloride was

synthesized, as reported earlier,¹⁴by first reducing 4-anisate methyl

ester with LiAl²H₄and then chlorinating the resulting 4-methoxy(1′,1′-

²H₂)benzyl alcohol with thionyl chloride. The identity and purity of

the alcohol and chloride were determined by NMR. 4-(²H₂)Methylanisole

was prepared by slow addition of 8.0 g (50.4 mmol) of the chloride to

a solution of 3.4 g of LiAlH₄in 100 mL of dry tetrahdyrofuran over a

period of 1 h. The reaction mixture was refluxed under dry Ar during

the addition period, and refluxing continued for another 3.4 h. Thin-

layer chromatography of a quenched aliquot of the reaction mixture

2

% H), Merck & Co. (Rahway, NJ); horse heart cytochrome c (type

VI), CH₃CH₂O²H (99.5+ atom % H), CH₃O²H (99.5+ atom % H),

phenazine methosulfate (PMS), and phenazine ethosulfate (PES), Sigma

Chemical Co. (St. Louis, MO); sodium 2,6-dichlorophenol indophenol

(DCIP), General Biochemicals (Chagrin Falls, OH). Solvents for high-

pressure liquid chromatography (HPLC) were HPLC grade. All other

chemical were of reagent grade.

2

1

Analytical Methods. Substrates purities were checked by (A) H

NMR using a Varian EM-360 spectrometer at room temperature

(chemical shifts, δ, in ppm relative to tetramethylsilane); (B) HPLC

(43) Koerber, S. C.; McIntire, W. S.; Bohmont, C.; Singer, T. P.

Biochemistry 1985, 24, 5276-5280.

(44) Wharton, D. C.; Tzagloff, A. Methods Enzymol. 1967, 10, 245-

250.

(42) Van Hook, W. A. Isotope Effects in Chemical Reactions; Collins,

C. J., Bowman, N. S., Eds.; Van Nostrand Reinhold: New York, 1979;

Chapter 1, pp 1-89.

5878 J. Am. Chem. Soc., Vol. 121, No. 25, 1999

McIntire et al.

indicated that the reaction had gone to completion. With stirring, 5 g

of Na₃PO₄‚12H₂O was very slowly added to the cooled mixture over

a 45-min period. The mixture was stirred overnight at room temperature,

then the thick gray slurry was dried in a rotary evaporator at 25 °C

and 20 mmHg, and finally 300 mL of H₂O was added. The aqueous

phase was extracted three times with 150-200 mL of CHCl₃. The

CHCl₃solution was dried over anhydrous MgSO₄and filtered, and

solvent was removed under reduced pressure. Yield: 6.0 g (48.4 mmol,

96.0%) of a light yellow liquid.

was further purified by sublimation as described for 4-ethylphenol. Mass

2

spectral analysis of deuterium content in the â-position: 95.7% H₃,

2

4.1% H₂, and 0.2% H₁, which corresponds to 98.5 atom % H.

4-Vinylphenol. Its synthesis was accomplished by published pro-

cedures.^45,46The material was pure as judged by HPLC (column 1; t_R

) 10.6 min; MeOH/H₂O, 1:9 (v/v) for 3 min then to 4:6 (v/v) in 3

min; 1 mL/min flow rate) and NMR in di(²H₃)methyl sulfoxide.⁴⁶It

had the same UV spectrum as commercial 4-vinylanisole in pure

MeOH.

The methyl ether of 4-(²H₂)methylanisole was cleaved with HBr

(49% in water) as described earlier for the synthesis of 4-(²H₃)-

methylphenol.¹⁴From 5.3 g of the anisole, 2.5 g of 4-(²H₂)methylphenol

and 2.4 g of 4-(²H₂)methylanisole were recovered by the following

procedure. The HBr solution was cooled, made alkaline (pH 12-14)

with NaOH, and extracted three times with 200 mL of CHCl₃(extract

1). The aqueous phase was adjusted to pH 7 with HCl, and the extraction

procedure was repeated (extract 2). Extract 1 was back-extracted three

times with H₂O and adjusted to pH 12 with NaOH, the aqueous phase

was neutralized with HCl and re-extracted three times with CHCl₃

(extract 3), and the combined organic phase was dried as before. Final

extract 1 contained 4-(²H₂)methylanisole and trace amounts of 4-(²H₂)-

methylphenol. Combined extracts 2 and 3 contained crude 4-(²H₂)-

methylphenol. 4-(²H₂)Methylphenol was purified in the same manner

as 4-(²H₃)methylphenol.¹⁴NMR (C²HCl₃): δ 2.17 (m, 1.0, C²H₂H),

6.02 (s, 1.2, OH), 6.70 and 6.96 (dd, 4.0, Ar-H). HPLC: t_R) 1.28

min (100%, column 3, H₂O/CH₃CN, 3:2, v/v). The material was further

purified by sublimation as described above for 4-methylphenol. Mass

R,S-(()-1-(4-Hydroxyphenyl)ethanol. Ten grams of 4-hydroxy-

acetophenone (73.5 mmol) was dissolved in 100 mL of H₂O/50 mL of

ethanol. With stirring, 5 g of solid NaBH₄was slowly added over a

20-min period to control foaming. After all of the NaBH₄was added,

the mixture was stirred until all bubbling ceased (∼1 h). The pH was

adjusted to 6-7 with H₃PO₄, and the mixture extracted three times

with 200 mL of water-saturated ethyl acetate. The solvent was removed

under reduced pressure. One hundred milliliters of dry ethyl acetate

was added to the residue, the mixture heated to boiling, and ethanol

added to dissolve all the solid. The solution was allowed to cool slowly

to room temperature, then to 0 °C, and finally at -20 °C. The resulting

crystals were filtered and washed with -20 °C ethyl acetate. The yield

of dried crystals was 3.9 g (28.3 mmol, 38.5%).

Other Substrates. The syntheses and purifications are reported

elsewhere.^46,47For R-(+)-1-(4-hydroxyphenyl)ethanol (all [R] values,

reported in degrees, were measured at 20 °C; C ) 5.00, in methanol),

[R]_D) +47.5, [R]₅₇₈) +49.7, [R]₅₄₆) + 56.9, [R]₄₃₆) +101.0,

[R]₃₆₅) +168.5; for S-(-)-1-(4-hydroxyphenyl)ethanol, [R]_D) -47.5,

[R]₅₇₈) -49.5, [R]₅₄₆) -56.9, [R]₄₃₆) -101.0, [R]₃₆₅) -168.4,

2

spectral analysis of deuterium content: 99.3% H₂and 0.7% H₁, for

2

a total of 99.7 atom % H.

2

the R,S-(()-1-(4-hydroxyphenyl)(1-²H₁)ethanol was 99.2 atom % H;

4-(²H₁)Methylphenol. Eight grams (0.051 mol) of the 4-methoxy-

benzyl chloride was reduced with LiAl²H₄, as described above for the

synthesis of 4-(²H₂)methoxyanisole using LiAlH₄. The yield was nearly

quantitative. Pure 4-(²H₁)methylphenol was obtained from 4-(²H₁)-

methylanisole as described in the previous section for the synthesis of

4-(²H₂)methylphenol. NMR (C²HCl₃): δ 2.20 (t, 2.0, C²HH₂), 6.11 (s,

1.3, OH), 6.68 and 6.96 (2d, 4.0, Ar-H). HPLC: R ) 1.31 min (100%,

column 3, H₂O/CH₃CN, 3:2, v/v). The material was further purified

by sublimation as described earlier for 4-methylphenol. Mass spectral

2

for R-(+)-1-(4-hydroxyphenyl)(1-²H₁)ethanol (99.0 atom % H), [R]_D

) +49.1, [R]₅₇₈) +51.4, [R]₅₄₆) +58.5, [R]₄₃₆) +103.7, [R]₃₆₅

)

+173.4; for S-(-)-1-(4-hydroxyphenyl)(1-²H₁)ethanol (99.0 atom %

²H), [R]_D) -48.8, [R]₅₇₈) -50.9, [R]₅₄₆) -58.3, [R]₄₃₆) -103.6,

[R]₃₆₅) -173.4, the R,S-(() -4-(1′-²H₁)ethylphenol was 97.1 atom %

²H; for R-(+)-4-(1′-²H₁)ethylphenol (96.7 atom % ²H), ([R] values were

measured at 20 °C; C ) 25.00, in ethanol) [R]_D) 0.16, [R]₅₇₈) 0.17,

[R]₅₄₆) 0.20, [R]₄₃₆) 0.37, [R]₃₆₅) 0.690); for S-(-)-4-(1′-²H₁)-

2

ethylphenol (98.3 atom % H for each of two preparations), [R]_D

)

analysis of deuterium content: 98.6% H.

-0.247 and -0.240, [R]₅₇₈) -0.242 and -0.260, [R]₅₄₆) -0.295

and -0.290, [R]₄₃₆) -0.563 and -0.505, [R]₃₆₅) -1.04 and -0.927.

Unfortunately, we could not devise a method, nor was one found in

the literature, for determining the exact enantiomeric purity of the R-

or S-isomer. For the purposes of this report, we assumed that the S-4-

ethylphenol preparation with the highest optical rotation was 100%

S-isomer, and this preparation was used for our kinetic studies. With

this assumption, we estimated the purity of the R-isomer to be 66% ee

(enantiomeric excess). All alcohols and all 4-ethylphenols were pure

as judged by HPLC using a 50- × 4.50-mm Hypersil 3-µm ODS

column: alcohols [including the R,S-(()-alcohol], t_R) 1.7 min, 0.8

mL/min flow rate, 7:3 H₂O/MeOH; 4-ethylphenols, t_R) 1.9 min, 1

mL/min flow rate, 45:55 H₂O/MeOH. Optical rotations were recorded

on a Perkin-Elmer model 141 polarimeter with a standard 1-dm,

temperature-thermostated cell.

4-(2′,2′,2′-²H₃)Ethylphenol. The 2′-hydrogens of 4-hydroxyac-

etophenone were exchanged by acid-catalyzed keto-enol tautomer-

ization. Six grams of 4-hydroxyacetophenone (0.176 mol exchangeable

hydrogens; three from the 2′-carbon, one from the phenolic hydroxyl

2

group) was dissolved in 10 mL of CH₃O²H, 4 mL of H₂O, and 200

µL of 35% ²HCl in ²H₂O and incubated at 40-50 °C for 16 h, protected

from atmospheric moisture. The liquid was removed by evaporation

under reduced pressure, and the process was repeated four more times.

This was followed by two more exchange steps using 10 mL of CH₃-

2

CH₂O²H and 0.4 mL of 35% HCl in H₂O. To remove all traces of

²HCl, the sample was dissolved in 5 mL of CH₃CH₂O²H, and the solvent

immediately evaporated. This step was repeated. The remaining solid

was then dissolved in 20 mL of CH₃CH₂OH to exchange deuterons at

the phenolic position with protons. The solvent was evaporated

immediately, and the procedure was repeated. The solid was completely

dried in a vacuum desiccator over P₂O₅. NMR analysis of the resulting

4-hydroxy(2′,2′,2′-²H₃)acetophenone [di(²H₃)methyl sulfoxide]: δ 2.46

(s, C²H₂H), 6.90 and 7.86 (dd, 4.0, Ar-H), 10.32 (s, 0.9, OH); estimated

Steady-State and Stopped-Flow Kinetic Assays with 1-(4-Hy-

droxyphenyl)ethanols. All steady-state kinetic assays were done in

50 mM Tris-HCl, pH 7.6, I ) 0.05 (KCl), at 25 °C, as described

earlier.¹⁴The electron-accepting substrate for PCMH was PES, and

the reactions were monitored at 600 nm, which measured the reduction

of DCIP (initial concentration, 95 µM) by reduced PES. Assays were

initiated by addition of enzyme to cuvettes containing buffer, phenolic

substrate, and dyes.

1

content of H in â-position, 1.6%.

Four grams of 4-hydroxy(2′,2′,2′-²H₃)acetophenone (0.0288 mol) was

reduced via the Clemmensen reaction in CH₃CH₂OH over Zn(Hg)

amalgam using HCl.¹⁴When the reaction was complete, 100 mL of

water was added, followed by addition of concentrated NaOH to

neutralize the mixture. The mixture was extracted three times with

CHCl₃, the combined organic phase dried with MgSO₄, and the solvent

removed under reduced pressure. The resulting viscous liquid was

purified by simultaneous distillation/sublimation.¹⁴This afforded 3.13

g of 4-ethylphenol-â-d₃as a white solid (25 mmol, 87% yield).

HPLC: t_R) 1.84 min (column 3, H₂O/CH₃CN, 3:2, v/v). The material

(45) Dale, W. J.; Hennis, H. E. J. Am. Chem. Soc. 1958, 80, 3645-

3649.

(46) Everhart, E. T.; Craig, J. C. J. Chem. Soc., Perkins Trans. 1 1991,

1701-1707.

(47) Everhart, E. T.; McIntire, W. S.; Craig, J. C., manuscript in

preparation.

Isotope Effects and PCMH

J. Am. Chem. Soc., Vol. 121, No. 25, 1999 5879

R-²H

Stopped-flow experiments were performed as described previ-

ously.^15,48The experiments were done in 50 mM Tris-HCl buffer

containing10 mM ^D-glucose, pH 7.6, I ) 0.05 (KCl), at 25 °C. The

solutions became anaerobic due to ther presence of catalase and glucose

oxidase in the buffer.⁴⁸

upper branch, Scheme 4

E + yxS {_{k /k}} ES_d

98 EP 9_k8 E + P (D removal)

k_2d

3

1

-1

lower branch, Scheme 4

Time Course Reactions. A typical reaction mixture contained 3-4

mM of the phenolic substrate, 30 µM horse heart cytochrome c, 1 mg

of total protein/mL of the crude cytochrome c oxidase, 40-70 µg/mL

of PCMH in 20-30 mL of 10 mM Tris-HCl buffer, pH 7.6, I ) 0.01.

At various times over the course of a reaction, 1-mL aliquots were

removed and quickly mixed with 0.15 mL of saturated (NH₄)₂SO₄to

quench the reaction. (It was found that cytochrome c is an excellent

electron acceptor at I e 0.01, but at I g 0.1, the reaction is halted.)

The quenched samples were centrifuged at 16000g for 3 min. The

proteins were separated from small molecules using Centricon-10

centrifuge filters; 30 min at 5000g at 4 °C. The filtered liquid was

analyzed directly by HPLC; for example, 5 µL was injected onto a

Burdick-Jackson C-18 column (0.4 × 25 cm; 5-µm particle size), and

the column was eluted with 2:3 (v/v) MeOH/H₂O at a 1 mL/min flow

rate, with 254- and 280-nm detection. The chromatograms were

recorded and peaks were integrated using an Altex model C-R1A

integrator (Beckman Instruments, Inc., Fullerton, CA). For preparative

runs, 1 mL was injected onto the same column, and the appropriate

fractions were collected. The MeOH was removed from these samples

using a stream of dry, filtered N₂. The remaining aqueous phase was

extracted four times with 0.25-0.5 mL of diethyl ether. The ether was

dried with anhydrous Na₂SO₄and the solvent removed in a stream of

N₂. Dried samples were taken up in 50 µL of dry MeOH and stored at

-70 °C. For analysis of the enantiomeric composition of the aryl

carbinols, a small volume of the sample collected directly from the

C-18 column (before MeOH evaporation and ether extraction) was

injected onto a Chiralcel OB column (cellulose tribenzoate-derivatized

macroporous silica gel, 0.4 × 25 cm, 5-µm particles; Daicel Chemical

Industries, Ltd., Japan) and eluted with n-hexane/2-propanol (various

mixtures from 7:1 to 19:1, v/v), with 254- and 280-nm detection.^24,25

This column gave nearly baseline separation of the R- and S- isomers

of the arylcarbinols. In early experiments, the enantiomeric composition

was determined using a Pirkle Type 1-A column (0.4 × 25 cm, 5-µm

particles; Regis Chemical Co., Morton Grove, IL).^22,23However, for

analyses on this column, the phenolic aryl carbinols had to be

diacetylated in order to separate the R- and S-isomers. Diacetylation

of the extracted and dried 1-mL aliquot samples was carried out with

0.1 mL of acetic anhydride and 0.1 mL of triethylamine at room

temperature for 2 h. The reagents were removed in a stream of dry N₂.

Samples were dissolved in 0.1 mL of n-hexane, and 20 µL was injected

onto the Pirkle column and eluted with n-hexane/2-propanol (various

ratios from 9:1 to 35:1, v/v), with 254- and 280-nm detection.

E + yxS {_{k /k}} ES_h9_k8 EP 98 E + P (H removal)

k_iii

ii

i

-i

S-²H

upper branch, Scheme 4

E + (1 - y)xS {_{k /k}} ES_h1

98 EP 9_k8 E + P (H removal)

k_2h

3

1

-1

lower branch, Scheme 4

E + (1 - y)xS {_{k /k}} ES_d1

98 EP 98 E + P (D removal)

k_iii

k_iid

i

-i

HH

upper branch, Scheme 4

E + (1 - x)S {

} ES_h2

8 EP 98 E + P (H removal)

k_2h2k₃

k₁/k_-1

lower branch, Scheme 4

E + (1 - x)S {_{k /k}} ES_h3

98 EP 98 E + P (H removal)

k_iii

k_iih

i

-i

In these reactions, it is assumed that the rate constants for binding and

catalysis are different for the upper and lower branches but the intrinsic

primary and secondary isotope effects are the same for both branches.

Therefore, k_2d) pk₂, k_ii) sꢀk₂, k_2h) sk₂, k_iid) pꢀk₂, k_2h2) k₂, k_iih

)

2

ꢀk₂, k_i) δk₁, k_-i) γk_-1, x ) fraction of H in substrate (97.6 atom

%), and y ) fraction R-²H isomer (83%).

d[E]/dt ) -k₁[S][E] - δk₁[S][E] + k_-1[ES_d] + γk_-1[ES_h] +

k_-1[ES_h1] + γk_-1[ES_d1] + k_-1[ES_h2] + γk_-1[ES_h3] +

k₃[EP] ) 0 (6)

d[ES_h]/dt ) yxk₁[S][E] - (k_-1+ pk₂)[ES_h] ) 0

d[ES_d]/dt ) yxδk₁[S][E] - (γk_-1+ sꢀk₂)[ES_d] ) 0

(7)

(8)

d[ES_h1]/dt ) (1 - y)xk₁[S][E] - (k_-1+ sk₂)[ES_h1] ) 0 (9)

d[ES_d1]/dt ) (1 - y)xδk₁[S][E] - (γk_-1+ pꢀk₂)[ES_d1] ) 0 (10)

d[ES_h2]/dt ) (1 - x)k₁[S][E] - (k_-1+ k₂)[ES_h2] ) 0 (11)

PCMH was anaerobically incubated with 4-vinylphenol or 1-(4-

hydroxyphenyl)ethanol (20 °C under Ar). At time zero, 30-mL reaction

mixtures contained 3.4 mM alcohol or 2.4 mM 4-vinylphenol, 0.11

µM glucose oxidase, 20 µg/mL catalase, 0.1 M ^D-glucose, 8.06 µg/mL

PCMH in 10 mM Tris-HCl, pH 7.6. For the 4-vinylphenol reaction, at

t ) 0, PCMH was reduced with a stoichiometric amount of 1-(4-

hydroxyphenyl)ethanol. At various times over a 28-50-h period,

aliquots were removed, processed, and analyzed by HPLC as described

in the previous paragraph.

d[ES_h3]/dt ) (1 - x)δk₁[S][E] - (γk_-1+ ꢀk₂)[ES_h3] ) 0

d[EP]/dt ) pk₂[ES_d] + ꢀsk₂[ES_h] + sk₂[ES_h1] + ꢀpk₂[ES_d1] +

(12)

Oxygen Electrode Assays. Assays for the enzymic oxidation of

4-ethylphenol were done at 25 °C in air-saturated 0.01 M Tris-HCl

buffer, pH 7.6. The reactions were monitored with a Clark electrode

using a Gilson Oxygraph equipped with a 1.6-mL reaction cell.²⁶In

addition to 4-ethylphenol and PCMH, the reaction mixture contained

horse cytochrome c and beef heart cytochrome c oxidase (vide supra).

k₂[ES_h2] + ꢀk₂[ES_h3] - k₃[EP] ) 0 (13)

[Et] ) [E] + [ES_h] + [ES_d] + [ES_h1] + [ES_d1] + [ES_h2] +

[ES_h3] + [EP] (14)

Derivation of Equations Used To Analyze Data: Derivation of

k

_cat/K Expressions. The example presented here is for the most

complicated case, that is, for (+)-R-4-(1-²H₁)ethylphenol, which is 83%

R-isomer and 97.6 atom % ²H. The reactions and equations given below

were derived using Scheme 4 in the text.

Since steady-state conditions are assumed, eqs 6-13 are set equal to

zero. These equations are a series of linear homogeneous differential

equations with regard to the enzyme species. [S] is assumed to be

constant. Of eqs 6-13, eq 13 can be considered redundant. From eqs

6-12 and 14, matrix Z is constructed (ref 27, pp 73-92):

(48) Ramsay, R. R.; Koerber, S. C.; Singer, T. P. Biochemistry 1987,

26, 3045-3050.

5880 J. Am. Chem. Soc., Vol. 121, No. 25, 1999

McIntire et al.

k_-1

-k_-1-pk₂

γk_-1

0

-γk_-1-ꢀsk₂

0

1

k_-1

γk_-1

0

k_-1

γk_-1

0

k₃

-k₁(1 +δ)[S]

xyk₁[S]

xyδk₁[S]

x(1-y)k₁[S]

x(1-y)δk₁[S]

(1-x)k₁[S]

(1-x)δk₁[S]

1

0

1

0

1

-k_-1-sk₂

Z )

-γk_-1-pꢀk₂

0

1

-k_-1-k₂

0

1

0

1

[

]

-γk_-1-ꢀk₂

1

ES_h

ES_d

ES_h1

ES_d1

ES_h2

ES_h3

E

EP

Next, a column vector, V, which is the solution to each of the differential

competitive inhibitor, and with unknown inhibitor, I, the following

equations apply:

equations, is constructed:

N_E

N

d[R]/dt ) k_catR/{1 + K_R/[R](1 + [S]/K_S)(1 + [P]/K_P)(1 +[I]/K_I)}

d[S]/dt ) k_catS/{1 + K_S/[S](1 + [S]/K_R)(1 + [P]/K_P)(1 +[I]/K_I)}

0

ES_h

N_S

d

N_S

0

h1

V )

(Z^-1)V )

N_S

From these equations, d[R]/d[S] ) (k_cat/K)_R[R]/{(k_cat/K)_S[S]} (ref 27,

pp 105-108), and d[R] ) (k_cat/K)_R[R]/{(k_cat/K)_S[S]}d[S]. Integration

of this equation from [R]₀to [R] and [S]₀to [S] yields ([R]/[R]₀)ⁿ)

[S]/[S]₀, where n ) (k_cat/K)_S/(k_cat/K)_R. Since the starting PCMH substrate

is racemic R,S-1-(4-hydroxyphenyl)ethanol, [R]₀) [S]₀. Thus, ([R]/

[R]₀)ⁿ) [S]/[R]₀) (T - [R])/[R]₀, where T is the total concentration

d1

h

0

[ ]

[

]

h3

E_t

N_EP

The steady-state velocity is defined as V ) k₃[EP]_ss, i.e., the rate of

product formation. To determine the steady-state concentrations of each

enzyme species, the following procedure is performed: (Z^-1)V, where

Z^-1is the inverse of Z. The resulting column vector is shown above.

[EP]_ssis defined as N_EP/∑N_i, where N_iare all the components of the

(Z^-1)V column vector. It turns out that the expression for (k_cat/K)_Ris

k₃(N_EP)/(N_E[S]). The expression for k_cat/K for unlabeled 4-ethylphenol

is determined in the same fashion. For all the deuterated 4-ethylphenols,

n-1

of alcohol at any time. Rearranging this equation gives T ) [R]ⁿ/[R]₀

+ [R].

Acknowledgment. This work was supported by a Depart-

ment of Veterans Affairs Merit Review grant, National Institutes

of Health Program Project Grant HL16251 (WSM), and the

University of California, San Francisco Mass Spectrometry

Facility (A. L. Burlingame, Director), which is supported by

the Biomedical Research Technology Program of the National

Center for Research Resources, NIH NCRR BRTP RR01614.

A preliminary account of some of this work was presented at

the Ninth International Symposium on Flavins and Flavopro-

teins, Atlanta, GA, June 7-12, 1987.²¹

k

_cat/K ) f(k₁,k_-1,k₂,k₃,s,p,A,x,y), where A ) δꢀ/γ. If it is assumed that

C ) k₂/k_-1) 0, it follows that k_-1. k₂. This assumption simplifies

the equations for k_cat/K for each deuterated and nondeuterated substrate.

k

_cat/K expressions for the deuterated 4-methylphenols were determined

in the same manner. The matrix algebra was done by symbolic

manipulation using MAPLE V, Release 4 software (Waterloo Maple

Software, Waterloo, Ontario, Canada).

Steady-State Enzyme Reaction with Two Competing Substrates.

With R and S as competing substrates, with P representing product as

JA984214G

Article Doi

A new procedure for deconvolution of inter-/intramolecular intrinsic primary and α-secondary deuterium isotope effects from enzyme steady-state kinetic data

DOI: 10.1021/ja984214g

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Article abstract of DOI:10.1021/ja984214g

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