Imino-oxy acetic acid dealkylation as evidence for an inner-sphere alcohol intermediate in the reaction catalyzed by peptidylglycine α-hydroxylating monooxygenase

Article abstract of DOI:10.1021/ja902716d

Peptidylglycine α-hydroxylating monooxygenase (PHM, EC 1.14.17.3) catalyzes the stereospecific hydroxylation of a glycyl α-carbon in a reaction that requires O₂ and ascorbate. Subsequent dealkylation of the α-hydroxyglycine by another enzyme, peptidylamidoglycolate lyase (PAL. EC 4.3.2.5), yields a bioactive amide and glyoxylate. PHM is a noncoupled, type II dicopper monooxygenase which activates O₂ at only a single copper atom, Cu_M. In this study, the PHM mechanism was probed using a non-natural substrate, benzaldehyde imino-oxy acetic acid (BIAA). PHM catalyzes the O-oxidative dealkylation of BIAA to benzaldoxime and glyoxylate with no involvement of PAL. The minimal kinetic mechanism for BIAA was shown to be steady-state ordered using primary deuterium kinetic isotope effects. The ^D(V/K)_{APPARENT, BIAA} decreased from 14.7 ± 1.0 as [O₂] → 0 to 1.0 ± 0.2 as [O₂] → ∞ suggesting the dissociation rate constant from the PHM·BIAA complex decreases as [O₂] increases; thereby, reducing the steady-state concentration of [PHM]_free. BIAA was further used to differentiate between potential oxidative Cu/O species using a QM/MM reaction coordinate simulation to determine which species could yield product O-dealkylation that matched our experimental data. The results of this study provided compelling evidence for the presence of a covalently linked Cu^II-alkoxide intermediate with a quartet spin state responsible BIAA oxidation.

Full text of DOI:10.1021/ja902716d

Published on Web 07/01/2009
Imino-oxy Acetic Acid Dealkylation as Evidence for an
Inner-Sphere Alcohol Intermediate in the Reaction Catalyzed
by Peptidylglycine r-Hydroxylating Monooxygenase
Neil R. McIntyre,† Edward W. Lowe, Jr.,‡ and David J. Merkler*
Department of Chemistry, UniVersity of South Florida, 4202 East Fowler AVenue,
Tampa, Florida 33620
Received April 5, 2009; E-mail: merkler@cas.usf.edu
Abstract: Peptidylglycine R-hydroxylating monooxygenase (PHM, EC 1.14.17.3) catalyzes the stereospecific
hydroxylation of a glycyl R-carbon in a reaction that requires O2 and ascorbate. Subsequent dealkylation
of the R-hydroxyglycine by another enzyme, peptidylamidoglycolate lyase (PAL. EC 4.3.2.5), yields a
bioactive amide and glyoxylate. PHM is a noncoupled, type II dicopper monooxygenase which activates
O2 at only a single copper atom, CuM. In this study, the PHM mechanism was probed using a non-natural
substrate, benzaldehyde imino-oxy acetic acid (BIAA). PHM catalyzes the O-oxidative dealkylation of BIAA
to benzaldoxime and glyoxylate with no involvement of PAL. The minimal kinetic mechanism for BIAA was
shown to be steady-state ordered using primary deuterium kinetic isotope effects. The D(V/K)APPARENT, BIAA
decreased from 14.7 ( 1.0 as [O2] f 0 to 1.0 ( 0.2 as [O2] f ∞ suggesting the dissociation rate constant
from the PHM ·BIAA complex decreases as [O2] increases; thereby, reducing the steady-state concentration
of [PHM]free. BIAA was further used to differentiate between potential oxidative Cu/O species using a QM/
MM reaction coordinate simulation to determine which species could yield product O-dealkylation that
matched our experimental data. The results of this study provided compelling evidence for the presence
of a covalently linked CuII-alkoxide intermediate with a quartet spin state responsible BIAA oxidation.
PHM and PAL are known, each coded by a distinct gene.3-5
Introduction
In the mollusk, Lymnae stagnalis, a pentafunctional enzyme is
produced with four PHM domains per PAL domain.6 The
complexities in the various forms of the PAM/PHM/PAL system
are not well understood, but hint at a novel regulatory mech-
anism for the in ViVo production of the amide and carbinolamide
products of this chemistry.
Of the two monofunctional enzymes in the PAM/PHM/PAL
system, PHM has been the most extensively studied, due to its
structural and mechanistic similarity to dopamine.
Peptidylglycine R-amidating monooxygenase (PAM, E.C.
1.14.17.3) catalyzes the final reaction in the biosynthesis of
R-amidated peptide hormones and may also have a role in the
production of oleamide and other bioactive primary fatty acid
amides.1,2 This chemistry is a two-step process with the
formation of an intermediate that has been stereospecifically
hydroxylated at the R-carbon of a glycine. PAM is bifunctional
consisting of two catalytic units: peptidylglycine R-hydroxy-
lating monooxygenase (PHM) and peptidylamidoglycolate lyase
(PAL). PHM catalyzes the glycine hydroxylation while PAL
catalyzes the carbinolamide dealkylation (Scheme 1a). The
relationship between PHM and PAL is multifaceted and species
specific. Mammalian PAM is coded for by a single gene;
alternative splicing of the PAM mRNA and proteolytic process-
ing of PAM yields a mixture of the bifunctional enzyme and
the monofunctional enzymes, PHM and PAL. In insects and
cnidarians, bifunctional PAM is not found; only monofunctional
ꢀ-monooxygenase (DꢀM).7 Both PHM and DꢀM catalyze
an O2-, copper-, and ascorbate-dependent hydrogen atom transfer
and substrate hydroxylation resulting in the stereospecific
hydroxylation of their respective substrates (Scheme 1a and 1b).
Both enzymes contain a solvent filled active site separating two
essential, noncoupled copper atoms.1,8-12 In PHM, the two
protein bound copper atoms have different roles in catalysis.
One copper atom, CuM, has two Nꢀ-histidines ligands (His240
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† Present address: Department of Molecular Medicine, College of
Medicine, University of South Florida, 12901 Bruce B. Downs Blvd.,
Tampa, FL 33612-4799.
(5) Hauser, F.; Williamson, M.; Grimmelikhuijzen, C. J. Biochem. Biophys.
Res. Commun. 1997, 241, 509–512.
‡ Present address: Vanderbilt University Center for Structural Biology,
465 21st Ave. South BIOSCI/MRBIII, Room 5144F, Nashville, TN 37232-
8725.
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Geraerts, W. P. FASEB J. 1999, 13, 735–748.
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10.1021/ja902716d CCC: $40.75  2009 American Chemical Society

Alcohol Intermediate in the Reaction Catalyzed by PHM
A R T I C L E S
Scheme 1. (a) Peptidylglycine R-Amidating Monooxygenase (PAM) and (b) Dopamine ꢀ-Monooxygenase (DꢀM) reactions
and His242) and a methionine sulfur ligand (Met314) and is
directly involved in dioxygen activation and substrate oxidation.
The other copper atom, CuH, has three Nδ-histidine ligands
(His107, His108, and His172) and is involved in electron transfer.
The PAL domain, unique to the PAM system, is zinc- and
calcium-dependent and catalyzes the dealkylation of the carbino-
lamide to the corresponding amide and glyoxylate (Scheme
1a).13,14
intramolecular electron transfer from the other Cu atom in the
active site, CuH, yielding a CuII-O• radical. Radical recombina-
tion of the substrate and Cu-O radical species produces an inner-
sphere alcohol intermediate. Subsequent hydrolysis of the inner-
sphere alcohol generates the hydroxylated product.
A highly reduced copper-oxo species is postulated for
substrate CR-H bond cleavage in both ‘copper-oxo’ mecha-
Intriguingly, DꢀM, without a PAL-like partner, will catalyze
oxidative dealkylation similar to the sequential reactions
catalyzed by PHM and PAL in bifunctional PAM.15 We report
herein the PAL-independent oxidative dealkylation of imino-
oxyacetic acids to the corresponding oximes and glyoxylate by
PHM. For PHM and DꢀM, the dealkylation reactions result
solely from the oxidation chemistry of their monooxygenase
domains. This similarity in dealkylation chemistry provides a
novel framework to study the mechanism of PAM catalysis.15-17
The role of oxygen activation in PHM and DꢀM has been
the subject of much study.18-22 The presumed nucleophile
responsible for hydrogen abstraction is predominantly considered
to be an end-on/η1 copper-superoxo radical species.11,23 Both
η1 and η2 copper-superoxo species had been prepared in models,
although none exhibited the propensity for both H-abstraction
and oxidation chemistry.24-29 Recently, the Karlin group
prepared end-on/η1 CuII-superoxo model complexes able to
orchestrate both H-abstraction and oxidation chemistry.30-33
Characterization of CuII-superoxo species was a critical advance;
however, ambiguities remain as to the identity of the actual
oxidant species.30
(10) Kulathila, R.; Consalvo, A. P.; Fitzpatrick, P. F.; Freeman, J. C.;
Snyder, L. M.; Villafranca, J. J.; Merkler, D. J. Arch. Biochem.
Biophys. 1994, 311, 191–195.
(11) Prigge, S. T.; Eipper, B. A.; Mains, R. E.; Amzel, L. M. Science 2004,
304, 864–867.
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L. M. Nat. Struct. Biol. 1999, 6, 976–983.
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Hand, T. A.; Mains, R. E.; Eipper, B. A. Biochemistry 2002, 41,
12384–12394.
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Chem. 2006, 281, 20873–20882.
(15) Padgette, S. R.; Wimalasena, K.; Herman, H. H.; Sirimanne, S. R.;
May, S. W. Biochemistry 1985, 24, 5826–5839.
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(18) Gherman, B. F.; Heppner, D. E.; Tolman, W. B.; Cramer, C. J. J. Biol.
Inorg. Chem. 2006, 11, 197–205.
(19) Chen, P.; Solomon, E. I. J. Am. Chem. Soc. 2004, 126, 4991–5000.
(20) Evans, J. P.; Ahn, K.; Klinman, J. P. J. Biol. Chem. 2003, 278, 49691–
49698.
(21) Itoh, S. Curr. Opin. Chem. Biol. 2006, 10, 115–122.
(22) Hatcher, L. Q.; Karlin, K. D. J. Biol. Inorg. Chem. 2004, 9, 669–683.
(23) Bollinger, J. M., Jr.; Krebs, C. Curr. Opin. Chem. Biol. 2007, 11,
151–158.
(24) Fujisawa, K.; Tanaka, M.; Moro-oka, Y.; Kitajima, N. J. Am. Chem.
Soc. 1994, 116, 12079–12080.
The two PHM mechanisms which involve a CuII-superoxo
nucleophile for substrate activation differ in the initial coordina-
tion geometry of the dioxygen species to copper (Scheme 2).
The ‘side-on/η2’ mechanism is supported by spectroscopic
evidence from side-on/η2 Cu/O species model studies and is
predicted to have an antiferromagnetically coupled singlet
ground state (see Scheme 3).25,34 When compared with the end-
on/η1 species, the side-on/η2 copperII-superoxo was calculated
to be thermodynamically preferred for H-transfer.19,35 Evidence
for the end-on/η1 species comes from the PHM crystal struc-
ture11 and the work of Blackburn et al.,36 suggesting that this
species is the nucleophile responsible for CR-H abstraction.
Both mechanisms include an end-on/η1 CuII-hydroperoxo species
following CR-H bond cleavage. The ‘side-on/η2’ mechanism
involves a direct hydroxylation of the CR-substrate radical,
through a ‘water-assisted’ radical recombination reaction result-
ing in the simultaneous reduction of the CuII-hydroperoxo and
CR-OH product release. Conversely, the CuII-hydroperoxo
species from the ‘end-on/η1’ mechanism is reduced via an
(25) Chen, P.; Root, D. E.; Campochiaro, C.; Fujisawa, K.; Solomon, E. I.
J. Am. Chem. Soc. 2003, 125, 466–474.
(26) Karlin, K. D.; Wei, N.; Jung, B.; Kaderli, S.; Zuberbuhler, A. D. J. Am.
Chem. Soc. 1991, 113, 5868–5870.
(27) Karlin, K. D.; Wei, N.; Jung, B.; Kaderli, S.; Niklaus, P.; Zuberbuhler,
A. D. J. Am. Chem. Soc. 1993, 115, 9506–9514.
(28) Karlin, K. D.; Kaderli, S.; Zuberbuhler, A. D. Acc. Chem. Res. 1997,
30, 139–147.
(29) Schatz, M.; Raab, V.; Foxon, S. P.; Brehm, G.; Schneider, S.; Reiher,
S.; Holthausen, M. C.; Sundermeyer, J.; Schindler, S. Angew. Chem.,
Int. Ed. 2004, 43, 4306–4363.
(30) Maiti, D.; Sarjeant, A. A.; Karlin, K. D. J. Am. Chem. Soc. 2007,
129, 6720–6721.
(31) Maiti, D.; Lucas, H. R.; Sarjeant, A. A.; Karlin, K. D. J. Am. Chem.
Soc. 2007, 129, 6998–6999.
(32) Lee, D. H.; Hatcher, L. Q.; Vance, M. A.; Sarangi, R.; Milligan, A. E.;
Sarjeant, A. A.; Incarvito, C. D.; Rheingold, A. L.; Hodgson, K. O.;
Hedman, B.; Solomon, E. I.; Karlin, K. D. Inorg. Chem. 2007.
(33) Maiti, D.; Fry, H. C.; Woertink, J. S.; Vance, M. A.; Solomon, E. I.;
Karlin, K. D. J. Am. Chem. Soc. 2007, 129, 264–265.
(34) Chen, P.; Solomon, E. I. J. Inorg. Biochem. 2002, 88, 368–374.
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13110.
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A R T I C L E S
McIntyre et al.
Scheme 2. Collection of Mechanisms Postulated for the Hydrogen Abstracting and Oxidation Reactions of Peptidylglycine R-Amidating
Monooxygenase (and Dopamine ꢀ-Monooxygenase), Respectively
nisms, which uncouple dioxygen reduction from substrate
activation. Hybrid QM/MM simulations suggest that a CuIII-
oxide/CuII-oxyl species is thermodynamically preferred relative
to CuII-superoxo or CuII-hydroperoxo nucleophiles.37-39 The
CuIII-oxide/CuII-oxyl species is analogous to the ferryl oxidant
(CpI) responsible for C-H activation in cytochrome P450.37,40
Both ‘copper-oxo’ mechanisms proceed from an initial end-on/
η1 CuII-superoxo species to a highly reduced copper-oxo species
by coupling intramolecular electron transfer with the acquisition
of two protons leading to water release. In ‘copper-oxo pathway
A’, the reduced copper-oxo species is CuII-oxyl with two
unpaired electrons ferromagnetically coupled to an unpaired
electron delocalized within the CuM domain yielding a quartet
spin state.37 Here, H-abstraction and hydroxylation are in concert
with spin inversion to a doublet ground state allowing substrate
(36) Bauman, A. T.; Yukl, E. T.; Alkevich, K.; McCormack, A. L.;
Blackburn, N. J. J. Biol. Chem. 2006, 281, 4190–4198.
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Alcohol Intermediate in the Reaction Catalyzed by PHM
A R T I C L E S
Scheme 3. Degenerate Electronic Ground-State Structures of the
CuII-Hydroperoxo and CuII-Hydroxyl Oxidant Species Proposed for
Direct Substrate Oxidation/Product Releasea
The hypothesis of the present study was that the PHM
catalyzed reaction proceeds through an ‘inner-sphere alcohol’
intermediate. To test this hypothesis we designed a novel PHM
substrate, benzaldehyde imino-oxy acetic acid (BIAA), which
would undergo PAL-independent dealkylation. Compared to
other known PHM substrates that release a hydroxylated
product,1 the unique ability of BIAA to decompose during
oxygen insertion allows isolation of the substrate oxidation step.
Characterization of BIAA dealkylation demonstrated that this
chemistry is exclusively dependent on PHM-catalyzed oxidation
(and independent of product release), allowing hybrid QM/MM
simulations of substrate radical oxidation to be accurately
modeled. Our data provides compelling evidence that the PHM
reaction does, in fact, proceed via an inner-sphere alcohol
intermediate.
Materials and Methods
Materials. Benzamide, R-hydroxyhippurate, benzaldoxime (BOX),
bromoacetic acid, [R-2H2]-bromoacetic acid, hydroxylamine hy-
drochloride, benzaldehyde, and rabbit muscle lactate dehydrogenase
were purchased from Sigma-Aldrich. Carboxymethylhydroxylamine
was purchased from TCI. Bovine liver catalase was purchased from
Worthington. N-dansyl-Tyr-Val-Gly was purchased from Fluka
Biochimika. Recombinant type A rat medullary thyroid carcinoma
bifunctional PAM was produced and purified as described42 and
was a gift from Unigene Laboratories, Inc. (Fairfield, NJ, see
www.unigene.com). All other reagents were of the highest quality
available from commercial suppliers.
a Please note, copper domain residues were omitted for clarity. Refer to
text for more detail.
oxidation and hydroxylated product release to be directly
coupled. In the ‘copper-oxo pathway B’, the CuII-oxyl species
is in a triplet ground state as the most thermodynamically
favorable species for H-abstraction. Spin inversion yields an
antiferromagnetically coupled singlet state to drive concerted
H-abstraction with substrate oxidation/product release. Simula-
tions by Crespo et al. favor the singlet state oxidant relative to
either quartet or triplet state oxidants.37
Synthesis of Benzaldehyde Imino-oxy Acetic Acid (BIAA).
Benzaldehyde was converted to the corresponding benzaldoxime
using a published procedure.43 Oxime (4.1 mmol) was dissolved
in 20 mL of H2O containing 5 equiv of NaOH, and the reaction
stirred at room temperature for 45 min, after which 1.5 equiv of
[R-H2]- or [R-2H2]-bromoacetic acid was slowly added in small
increments with continued stirring. Upon completion (as determined
by TLC using a 1:3 hexanes/ethyl acetate as the mobile phase) the
reaction was acidified with dilute HCl, the BIAA was collected in
a Bu¨chner funnel, and solvent was removed by washing the solid
with petroleum ether. BIAA was recrystallized twice with benzene/
petroleum ether, yielding a white crystalline solid. Compound purity
Analysis of the four mechanisms outlined in Scheme 2 shows
commonality between the ‘side-on/η2,35,41 and both ‘copper-
oxo’38,39 mechanisms. Product release and substrate oxidation
are simultaneous, lacking covalent interaction between H-donor
and Cu/O acceptor (‘free-product’). The direct hydroxyl transfer
reactions of the ‘side-on/η2’ and ‘copper-oxo pathway B’
mechanisms are electronically similar as hydroxyl transfer
begins with an antiferromagnetically coupled singlet state.
Therefore, as the reaction coordinate proceeds toward substrate
oxidation/product release, the transition states of both ‘copper-
oxo pathway B’ and ‘side-on/η2’ species become superimposable.
Conversely, the ‘end-on/η1’ mechanism is defined by an
inner-sphere alcohol intermediate produced through a radical
recombination reaction.7,20 The CuII-oxyl radical species in the
‘end-on/η1′ mechanism is unique in that the reductive cleavage
of O-O bond in the CuII-hydroperoxo moiety results from an
intramolecular reduction followed by homolysis to yield the
active species. Unlike the ferryl (FeIVdO; CpI) complex of
P450, the CuII-oxyl lacks the strong electron delocalization into
the d-orbitals of the metal, allowing full radical stabilization in
the oxygen 2π* orbital.41 Formation of the CuII-oxyl species
by reduction of the CuII-hydroperoxo followed by homolytic
O-O bond cleavage suggests that this cupryl species must exist
as a quartet, with well-defined radical character on the oxygen.
1
was analyzed by RP-HPLC and H and 13C NMR.
1
[R-H2]-BIAA: H NMR analysis (250 MHz, MeOD-d4) δ 4.96
(singlet, 2H, CH2, R-methylene), 7.66-7.87 (m, 5H, ArH), 8.48
(singlet, 1H, H-CdN). 13C NMR analysis (62.5 MHz, MeOD-d4)
δ 171.841 (CdO, carboxylic acid), 149.486 (CdN, imine), 130.911
(Ar, C-1), 129.188 (Ar, C-4), 127.673 (Ar, C-3, C-5), δ126.136
(Ar, C-2, C-6), δ69.325 (CH2, R-methylene), mp 93-94 °C.
[R-2H2]-BIAA: 1H NMR analysis (400 MHz, Me2SO-d6): δ
7.397-7.589 (m, 5H, ArH), 8.302 (singlet, 1H, H-CdN). 13C
NMR analysis (100 MHz, Me2SO-d6) δ 171.660 (CdO, carboxylic
acid), 150.455 (CdN, imine), 130.865 (Ar, C-1), δ129.502 (Ar,
C-4), 127.688 (Ar, C-3, C-5), 125.644 (Ar, C-2, C-6), mp 96-97
°C.
Standard PAM Reaction Conditions. All reactions were
performed at 37.0 ( 0.1 °C in the following solution: 100 mM
MES/NaOH (pH 6.0), 30 mM NaCl, 1% (v/v) EtOH, 0.001%
(v/v) Triton X-100, 1.0 µM Cu(NO3)2, 5.0 mM sodium ascorbate,
and 5.75 µg/mL bovine liver catalase, 217 µM O2 and the desired
oxidizable substrate. Morrison and Billett44 showed that the ambient
O2 concentration at 1 atm and 37 °C is 217 µM.
(37) Crespo, A.; Marti, M. A.; Roitberg, A. E.; Amzel, L. M.; Estrin, D. A.
J. Am. Chem. Soc. 2006, 128, 12817–12828.
(38) Kamachi, T.; Kihara, N.; Shiota, Y.; Yoshizawa, K. Inorg. Chem. 2005,
44, 4226–4236.
(39) Yoshizawa, K.; Kihara, N.; Kamachi, T.; Shiota, Y. Inorg. Chem. 2006,
45, 3034–3041.
(42) Merkler, D. J.; Young, S. D. Arch. Biochem. Biophys. 1991, 289, 192–
196.
(40) Roth, J. P. Curr. Opin. Chem. Biol. 2007.
(41) Chen, P.; Bell, J.; Eipper, B. A.; Solomon, E. I. Biochemistry 2004,
43, 5735–5747.
(43) Liu, K.-C.; Shelton, B. R.; Howe, R. K. J. Org. Chem. 1980, 45, 3917–
1919.
(44) Morrison, T. J.; Billett, F. J. Chem. Soc. 1948, 2033–2035.
9
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A R T I C L E S
McIntyre et al.
Reverse-Phase HPLC (RP-HPLC) Analysis of PAM-Cata-
lyzed Reactions. A RP-HPLC separation for N-benzoylglycine,
R-hydroxy-N-benzoylglycine, benzamide, BIAA, and BOX was
performed at 50 °C using a 65% (v/v) 50 mM potassium phosphate
pH 6.0, 30% (v/v) methanol, and 5% (v/v) acetonitrile with a flow
rate of 1.0 mL/min. Analytes were detected at 248 nm (Figure A,
Supporting Information). A Hewlett-Packard HP-1100 HPLC
equipped with a Phenomenex C18-Hypersil column (100 mm × 4.6
mm) attached to a HP-variable wavelength detector was employed
for all HPLC separations. Standard curves for N-benzoylglycine,
R-hydroxy-N-benzoylglycine, benzamide, BIAA, and BOX were
prepared from the corresponding peak area values.
Characterization of Oxidation Product from BIAA. PAM (360
nM) was added to initiate the oxidation of 4.0 mM BIAA under
standard reaction conditions, reaction volume ) 0.5 mL. After a
20 h incubation, the reaction was diluted by addition of 8 mL of
H2O, extracted with ethyl acetate (20 mL × 3), rinsed with brine,
and dried over MgSO4. The product was isolated by flash column
chromatography and concentrated under reduced pressure. A small
fraction of the BIAA oxidation product was first evaluated by RP-
HPLC by retention time comparison to commercially available
benzaldoxime. The remainder of the BIAA oxidation product was
analyzed by 13C NMR using a Bruker 250 MHz NMR.
both substrates under standard reaction conditions. Variation in
initial [O2] was accomplished by mixing different proportions of
an O2 and N2 with the final [O2] was calibrated versus 217 µM
[O2] at 37.0 ( 0.1 °C after a 4 min equilibration.48 Enzyme-
dependent rates of O2 consumption were obtained after initiation
of the reaction with a ∼110 nM (4-5 µL) of PAM. Background
rates of O2 consumption obtained in the absence of PAM were
subtracted from the enzyme-dependent rates. Values for both
D(VMAX/KM)APPARENT,
and D(VMAX/KM)APPARENT,
were
BIAA
OXYGEN
attained from the ratio of protio/deuterio kinetic parameters (eq 1)
using Kaleidagraph.
VMAX,APPARENT*[S]
rate )
(1)
KM,APPARENT + [S]
Values for the D(VMAX/KM)APPARENT,
were also determined
BIAA
directly at a single BIAA concentration below the KM,APPARENT
values for [O2], as the observed rate/[substrate] at low substrate
concentration is expressed in the units of a second order rate
constant, mM-1 s-1 (eq 2). The D(VMAX/KM)APPARENT,BIAA was
calculated by dividing the (VMAX/KM)APPARENT,BIAA values obtained
for protiated substrate by the deuterated substrate. The reported
error is the standard error.
Determination of Glyoxylate Stoichiometry. The stoichiometry
of BIAA oxidation to glyoxylate formation was determined under
standard reaction conditions using 5.0 mM BIAA as the oxidizable
substrate. BIAA oxidation was initiated by the addition of 80 nM
PAM. At the desired time, a 40 µL reaction aliquot was removed
and the reaction terminated by the addition of trifluoroacetic acid
(TFA) yielding a final concentration of 1% (v/v) TFA. Conversion
of BIAA to benzaldoxime was monitored by RP-HPLC. All the
RP-HPLC samples were diluted to a constant BOX concentration
of 48 µM (midpoint of the BOX standard curve) for glyoxylate
analysis. The concentration of glyoxylate in the acid-quenched
samples was determined by the lactate dehydrogenase-mediated
reduction of glyoxylate to glycolate coupled to the oxidation of
NADH to NAD+ (∆ε340 ) 6.22 × 103 M-1 cm-1). Glyoxylate-
containing samples were combined with 7.6 units/mL lactate
dehydrogenase in 100 mM potassium phosphate pH 7.0 and 220
µM NADH and incubated for 1 h. at 37 ( 0.1 °C. The increase in
absorbance at 340 nm was measured using a JASCO V-530 UV-vis
spectrophotometer.
Vmax,APPARENT*[S]
rate )
f rate )
KM,APPARENT + [S]
Vmax,APPARENT*[S]
KM,APPARENT
rate
, as [S] , KM,APPARENT
VMAX,APPARENT
∴
≈
≡ s-1 mM-1 (2)
[S]
KM,APPARENT
The plot of D(VMAX/KM)APPARENT,
versus [O2] was fit to a
BIAA
hyperbolic curve and analyzed by the least-squares method, while
the D(VMAX/KM)APPARENT,
versus [BIAA] was described as
OXYGEN
the mean isotope effect ( standard deviation. The full initial rate
data matrix for both R-diprotiated and R-dideuterated substrates
versus [O2] concentration were analyzed separately and fit to both
eq 3 (steady-state preferred) and eq 4 (equilibrium preferred) using
the programs of Cleland.49 Single point (VMAX/KM)APPARENT, BIAA
D
determinations were excluded from this analysis
Determination of Oxygen Stoichiometry during BIAA Oxi-
dation. Amperometric analysis was used to correlate total [O2]
consumed during PAM catalysis under conditions of limiting
[BIAA]. The PAM-dependent consumption of O2 was followed
using a Yellow Springs Instrument model 53 oxygen monitor
equipped with a polarographic oxygen electrode interfaced with a
personal computer using a Dataq Instruments analogue/digital
converter (model DI-154RS) as previously described in McIntyre
et al.45
rate )
Vmax[BIAA][O2]
KD,BIAAKM,O + KM,BIAA[O2] + KM,O [BIAA] + [BIAA][O2]
2
2
(3)
VMAX[BIAA][O2]
rate )
KD,BIAAKM,O + KM,O [BIAA] + [BIAA][O2]
2
2
Background levels of [O2] consumed, obtained in the absence
of enzyme, were subtracted from the total [O2] after the addition
of PAM.46,47 Standard reactions were initiated with 2.0 nM PAM
with the BIAA concentrations being 20, 35, 75, or 100 µM. For
each initial [BIAA], the concentration of O2 (217 µM) was in exces,
and thus, BIAA was the limiting reagent. The total [O2] consumed
at each initial [BIAA] was determined from the asymptote of the
O2 consumption progress curve using SigmaPlot 8.0.
Measurement of Noncompetitive Kinetic Isotope Effects.
Initial rates of [R-H2]- and [R-2H2]-BIAA were compared indepen-
dently as a function of initial [O2]. Working stocks of BIAA were
calibrated by correlating the O2 consumption to 20 µM BIAA for
(4)
Rates from eqs 3 and 4 are composed of the substrate concentra-
tions; BIAA and O2, the maximal initial velocity; VMAX, Michaelis
constants; KM,BIAA and KM,O2, and the dissociation constant for
BIAA, KD,BIAA. Further analysis of D(VMAX/KM)APPARENT
, BIAA vs [O2]
and D(VMAX/KM)APPARENT,
vs [BIAA] data were performed
OXYGEN
to extrapolate values for D(VMAX/KM)APPARENT, OXYGEN, as [O2] f 0
µM and the value of D(VMAX/KM)APPARENT, OXYGEN, as [O2] f ∞ µM.
The D(VMAX/KM)
as [O2] f 0 µM values were
APPARENT, OXYGEN
determined through nonlinear regression analysis fitting replot data
to eq 5. The values for D(VMAX/KM)APPARENT,OXYGEN as [O2] f ∞
µM were calculated from the reciprocal expression of eq 5, shown
with the expression in eq 6.50
(45) McIntyre, N. R.; Lowe, E. W., Jr.; Chew, G. H.; Owen, T. C.; Merkler,
D. J. FEBS Lett. 2006, 580, 521–532.
(46) Buettner, G. R.; Jurkiewicz, B. A. Radiat. Res. 1996, 145, 532–41.
(47) Samuni, A.; Aronovitch, J.; Godinger, D.; Chevion, M.; Czapski, G.
Eur. J. Biochem. 1983, 137, 119–24.
(48) Francisco, W. A.; Merkler, D. J.; Blackburn, N. J.; Klinman, J. P.
Biochemistry 1998, 37, 8244–8252.
(49) Cleland, W. W. Methods Enzymol. 1979, 63, 103–138.
9
10312 J. AM. CHEM. SOC. VOL. 131, NO. 29, 2009

Alcohol Intermediate in the Reaction Catalyzed by PHM
A R T I C L E S
coordinate calculations. For the QM portion of the calculation a
spin unrestricted hybrid density functional theory, with B3LYP
hybrid-exchange functional using an LAVCP* basis set was used
to more accurately define copper atoms using effective core
potentials.54-57 The quantum mechanical region was treated with
spin unrestricted open-shell calculations (UDFT) due to the presence
of a radical in the system The molecular mechanics portion of the
protein was treated with the OPLS_2001 force field of Jorgensen.58,59
There were two species of interest for study in the PHM oxidation
reaction: The copper-hydroxyl (CuII-OH) and the copper-oxyl
radical (CuII-O•). The CuII-superoxo species and the bound ligand
were set to CuII-OH or CuII-O• and BIAA R-carbon radical in the
input file. The quantum mechanical (QM) region was calculated
using the CuM atom and domain residues (His240, His242, Met314),
the metal-bound oxygen species (either OH or OO•), and BIAA
CR radical substrate. On this selection, a Q-site job was run without
coordinate constraints to determine the optimized starting point
geometry. The calculation was repeated with the oxygen species
frozen and the CR of BIAA frozen to determine the energetic
minima starting point. Analysis of the reaction coordinate for
substrate oxidation was determined through the difference calculated
from incremental increase in the distance between the copper bound
oxygen bond and the corresponding decrease in the CR-O(H) bond.
The coordinates determined for each change in reaction coordinate
were refrozen for those two atoms and the energy calculated
quantum mechanically. Results were visualized using the Maestro.60
k3[O2]
k5H 1 +
(
)
k2
k3[O2]
k2
k4 + k5H 1 +
(
)
Vmax
KM
k5H
k5D
D
)
+
(
)
k3[O2]
APPARENT,BIAA
k5H 1 +
(
)
k2
1 +
k3[O2]
k4 + k5H 1 +
(
)
k2
(5)
1
)
VMAX
KM
VMAX
D
- D
(
)
(
)
KM
[O2]fOµM
1
K
O2
+ 1 K )
(
)
VMAX
KM
VMAX
KM
D
- D
(
)
(
)
[O2]f∞
[O2]fOµM
k2(k4 + k5 + k5H)
k3(k5 + k5H)
(6)
Quantum Mechanical/Molecular Mechanics Reaction Co-
ordinate. The crystal structure of reduced peptidylglycine R-hy-
droxylating monooxygenase (PHM) was acquired from the protein
databank (1SDW, 1.85 Å). All waters were removed from the
structure along with all occurrences of the cocrystallized peptide
ligand (IYT) outside of the active site.11,12,51 The bond orders and
charges were then corrected on the cocrystallized protein structure.
The copper atoms “atom types” were designated as ‘CuI’, hydrogens
were added, and a restrained minimization was performed. The
bounding box containing all ligand atoms was set to 14 Å3.
Molecular oxygen was removed to prepare the structure using the
ProteinPrep wizard for the quantum polarized ligand docking of
BIAA (QPLD; Schro¨dinger First Discovery Suites, www.
schrodinger.com). Initially, Glide52 was used to select five top poses
using standard precision (SP) mode. These ligand-receptor com-
plexes were analyzed using the Q-site53 module where the bound
ligand for each selected pose was treated by ab initio methods to
calculate partial atomic charges utilizing electrostatic potential fitting
within the receptor. All poses exceeding root mean squared (rms)
and maximum atomic displacement of value 0.5 and 1.3 Å were
rejected. Single-point energy determination was treated by the
QPLD algorithm to determine the most energetically favorable
ligand pose with respect to the receptor. Glide was then used to
redock the ligand using each of the ligand charge sets previously
calculated in Q-site.53 To the QPLD output pose with the BIAA
substrate docked into the PHM crystal structure, 3 Na+ ions were
added to neutralize (zero net charge) the system along with the
molecular oxygen using the coordinates from the original pdb. The
system was explicitly solvated using the TIP3P water model to 10
Å from the receptor and equilibrated.37 It should be noted that the
O2 coordinates were frozen during the entire system equilibration
because it was not formally bound to the CuM domain and would,
therefore, traverse the active site during equilibration if its
coordinates were not restrained. The final minimized structure of
the PHM-ligand central complex was attained through quantum
mechanical treatment of the CuM pocket while treating the remainder
of the protein structure classically to yield the correct final geometry.
The resulting structure was used with Q-Site53 for reaction
Results
Reaction Stoichiometry and Product Identification for the
PHM-Catalyzed Oxidation of BIAA. The 13C NMR of the PHM-
generated product generated from BIAA exhibited a spectrum
with chemical shifts similar to those of the BIAA starting
material, containing signature aromatic and imine carbon shifts
(represented A-E, Figure B, Supporting Information). However,
the two carbon shifts found at 171.8 and 69.3 ppm from the
O-acetyl moiety in BIAA were not present in the product
spectrum. The RP-HPLC retention time of the isolated product
was 12.4 min, identical within experimental error to that of
commercially available benzaldoxime, and differed significantly
from that of the BIAA starting material (6.0 min).
Using BIAA as the limiting substrate, the stoichiometry for
the PHM-dependent O2 consumption was 0.97 ( 0.06 O2
consumed/BIAA oxidized. (Figure C and Table C Supporting
Information). The LDH-catalyzed reduction of glyoxylate to
glycolate with the concomitant oxidation of NADH to NAD+
provided a convenient spectroscopic method (a) to demonstrate
that glyoxylate is a product of the PHM-catalyzed oxidation of
BIAA and (b) to measure the glyoxylate concentration generated
from BIAA upon PHM treatment. Percent conversion of BIAA
to BOX was measured by RP-HPLC. Using the individual
methods to measure [BOX] and [glyoxylate], we determined
that the [glyoxylate]/[BOX] stoichiometry for the PHM-
catalyzed oxidation of BIAA was 1.1 ( 0.1 (Figure 1).
Stability of the BIAA Oxidation Product. Following the
PHM-dependent oxidation of BIAA, there are two possible
routes for the generation of the observed products, BOX and
(54) Becke, A. D. J. Chem. Phys. 1993, 98, 1372–1377.
(55) Becke, A. D. J. Chem. Phys. 1993, 98, 5648–5652.
(56) Hay, J. P.; Wadt, W. R. J. Chem. Phys. 1985, 82, 299–310.
(57) Cui, Q.; Elstner, M.; Kaxiras, E.; Frauenheim, T.; Karplus, M. J. Phys.
Chem. B 2001, 105, 569–585.
(50) Klinman, J. P.; Humphries, H.; Voet, J. G. J. Biol. Chem. 1980, 255,
11648–11651.
(51) Prigge, S. T.; Kolhekar, A. S.; Eipper, B. A.; Mains, R. E.; Amzel,
L. M. Science 1997, 278, 1300–1305.
(58) Jorgensen, W. L.; Tirado-Rives, J. J. Am. Chem. Soc. 1988, 110, 1657–
1666.
(59) Kaminski, G. A.; Friesner, R. A.; Tirado-Rives, J.; Jorgensen, W. L.
J. Phys. Chem. B 2001, 105, 6474–6487.
(52) GLIDE Schrodinger, LLC Portland, OR, 2000.
(53) QSITE Schrodinger, LLC Portland, OR, 2000.
(60) MAESTRO Schrodinger, LLC Portland, OR, 2002.
9
J. AM. CHEM. SOC. VOL. 131, NO. 29, 2009 10313

A R T I C L E S
McIntyre et al.
D
The extrapolated value of (V/K)APPARENT, BIAA as [O2] f 0 was
determined to be 14.7 ( 1.0 (Figure D, Supporting Information),
and the D(V/K)APPARENT, BIAA as [O2] f ∞ was calculated to be to
1.01 ( 0.03 (Figure E, Supporting Information). The value for
the D(V/K)APPARENT, O2 based on a steady-state preferred mechanism
(eq 3) was calculated to be 5.8 ( 0.7 (Table 1). This value is
consistent with experimental determinations of D(V/K)APPARENT,O2
obtained at different initial O2 concentrations at one fixed initial
BIAA concentration, yielding an average D(V/K)APPARENT, O2 of 4.7
( 1.1 (Figure 4, blue circles, O). D(VMAX) was also calculated to
be near unity at 0.8 ( 0.1, while D(KD,BIAA) was found to be 2.1 (
0.5. The KM,BIAA parameters for both R-protiated and R-dideuterated
BIAA were determined to be negative with large standard errors.
This suggests that the KM,BIAA was not statistically significant,
consistent with a steady-state preferred kinetic mechanism.48,49
Figure 1. Determination of the PAM-dependent ratio of formed products
(oxime and glyoxylate) using the imino-oxy acetic acid substrate. The
concentrations of oxime and glyoxylate products were independently
determined by RP-HPLC and spectrophotometry.
QM/MM Reaction Coordinate. The two species evaluated
computationally were the CuII-OH (singlet) and the CuII-O•
(quartet). The potential energy of the reaction coordinate for
the CuII-OH species, calculated as the difference in bond distance
between the CR-O and Cu-OH bonds, displayed a nearly linear
increase in relative energy from the starting point to a ‘saddle
point’ value of +45.93 kcal/mol. Following this point, the
calculated energy of the reaction coordinate decreased to -25.98
kcal/mol (Figure 5). The CR r O bond lengths decreased from
4.60 to 2.80 Å at the energetic ‘saddle point’, resting at a final
distance of 1.30 Å. The CuII f OH bond lengths increased over
the reaction coordinate from 1.82 through 2.81 Å at the energetic
maxima, to 4.08 Å at the energetic minima. The imino-oxy T
R-carbon bond lengths for BIAA increased 0.16 Å over the
reaction coordinate, from 1.36 to 1.52 Å (Figure 5). The
sequence shown in Scheme B and movie A (Supporting
Information) represent the quantum mechanical aspects of the
performed QM/MM study. The displayed poses correspond
directly with their respective steps in Figure 5. The orientation
of the carboxyl of BIAA was unchanged until the step prior to
the saddle point (+37.45 kcal/mol). Following this point, a
coupled rotation in the hydroxyl moiety and the carboxyl of
BIAA was shown which oriented the R-carbon essentially
perpendicular to the approaching hydroxyl group (Scheme B,
Supporting Information).
The reaction coordinate for the copper-alkoxide (CuII-O•)
species (Figure 6) displayed a more complex relationship with
potential energy trend, relative to that of CuII-OH, as an energetic
plateau was observed midway to the saddle point at ∼28 kcal/
mol. Following this, potential energy at the ‘saddle point’ was
+54.75 kcal/mol, +12.82 kcal/mol greater than that observed
for the reaction coordinate for the CuII-OH species. Although a
higher potential energy was observed for the CuII-O• species, a
rapid decline in the potential energy was observed beyond the
‘saddle point’ on the reaction coordinate (Figure 6), in contrast
to slow decline observed for the CuII-OH species (Figure 5).
The ‘saddle points’ for the CuII-OH species occurred with nearly
equal bond lengths for the OH between the CR and CuII (∼2.8
Å at RC ) 0.014). Conversely, the ‘saddle point’ of the CuII-
O• reaction coordinate was much less symmetrical, reaching
its maxima when the CR-O and CuII-O bond lengths were 1.9
and 3.7 Å, respectively. The CuII-OH potential energy decreased
over the reaction coordinate (RC) from +45.93 kcal/mol at RC
) 0.014 to -25.97 kcal/mol at RC ) 2.78 while the CuII-O•
decreased from +58.75 kcal/mol at RC ) 1.78 to -9.68 kcal/
mol at RC ) 3.05. Therefore, the total change in potential
energy for each species was similar for each species (within
glyoxylate (Scheme 4a). Path A in Scheme 4 represents a stable
R-hydroxylated BIAA which then requires PAL to catalyze
BOX and glyoxylate formation while path B requires only PHM
for BOX and glyoxylate formation from BIAA. Here, PHM-
catalyzed oxidation of the R-carbon would accompany a PAL-
independent, chemical dealkylation to yield BOX and glyoxylate.
Our approach to differentiating between paths A and B in
Scheme 4 was to measure BOX formation in the presence of
R-hydroxyhippurate, a known PAL substrate.17,48 If the putative
R-hydroxylated BIAA product generated by PHM requires PAL
for dealkylation, BOX formation would be inhibited by R-hy-
droxyhippurate. As a control, benzamide formation via the PAL-
catalyzed dealkylation of R-hydroxyhippurate would also be
inhibited by BIAA. Of course, if the BIAA product generated
by PHM is not a PAL substrate, neither BOX formation nor
benzamide formation would be affected.
The progress curves for BOX formation from BIAA measured
in the presence of either 6 mM or 10 mM R-hydroxyhippurate
were unaffected at all BIAA concentrations employed (Figure
2). Similarly, the progress curves for benzamide formation from
R-hydroxyhippurate were unaffected by the presence of 0.5, 2,
or 3 mM BIAA (Figure 3).
Kinetic Isotope Effects. The D(V/K)APPARENT,BIAA decreased as
the concentration of O2 decreased, while the D(V/K)APPARENT,O2
remained constant over the range of BIAA concentrations used
(Tables A and B, Supporting Information). Data fit to the steady-
state preferred mechanism (eq 3) had root square values (σ) of
0.237 and 0.140 for R-protiated and R-dideuterated BIAA,
respectively. Conversely, data fit to the equilibrium preferred
mechanism (eq 4) displayed root square values (σ) of 0.236
and 0.156 for the protiated and R-dideuterated substrates,
respectively. An approximate 10-fold increase in variance was
observed for the fit to equations for the equilibrium preferred
mechanism, as compared with its steady-state preferred coun-
terpart.49 A large increase in the average residual least-squares
(variance) term was observed, showing a greater deviation in
error between experimental rates (VEXP) and the predicted/
optimized rates (VOPT) for the linear regression model. The
variance for [R-1H2]-BIAA and [R-2H2]-BIAA increased from
0.0561(H) and 0.0230(D) from for steady-state preferred to
0.555(H) and 0.242 (D) to for equilibrium preferred. Kinetic
data fit to the steady-state preferred model (eq 3) displayed an
increased statistical significance based on their variance terms,
though the square root (σ) values were statistically very similar.
The graphs of 1/rate vs 1/[BIAA] and 1/rate vs 1/[O2] both
exhibit intersections in the same quadrant, with a negative abscissa
and positive ordinate when fit to the steady-state preferred equation.
9
10314 J. AM. CHEM. SOC. VOL. 131, NO. 29, 2009

Alcohol Intermediate in the Reaction Catalyzed by PHM
A R T I C L E S
Figure 2. Time course for the PAM-dependent conversion of 3, 2, and 0.5 mM benzaldehyde imino-oxy acetic acid to benzaldoxime in the presence and
absence of R-hydroxy-N-benzoylglycine. Results were calculated from RP-HPLC analysis (percent conversion).
Figure 3. Resolved time course for the conversion of 6 mM and 10 mM R-hydroxy-N-benzoylglycine to benzamide in the presence and absence of 0.5, 2,
and 3 mM BIAA (BenIAA). Please note, time point data were truncated for clarity.
Scheme 4. (a) Representation of the Two Possible Pathways for BOX and Glyoxylate Formation Following BIAA Oxidation in the
PHM-Domain. Please Note That ‘asc’ and ‘sda’ Represent Single Electron Contributions from Exogenous Ascorbate and
Semi-dehydroascorbate; (b) Experimental Design to Kinetically Differentiate the Two Possible Degradation Pathways for BIAA to Its
Corresponding BOX Product Following PHM-Dependent Oxidation
experimental error), 68.43 kcal/mol for CuII-O• and 71.90 kcal/
mol for CuII-OH.
Substrate Dealkylation. The increase in imino-oxy T R-car-
bon bond length for the CuII-O• species (Figure 6) was observed
to be constant over the reaction coordinate until the energetic
‘saddle point’ was reached. It was here that the substrate bond
length was dramatically increased as compared to the effect
observed studying the CuII-OH species, ranging from 1.42 to
2.91 Å. The corresponding poses for the quantum mechanically
treated region of the CuII-OH simulation showed very little
change in substrate orientation, with major functionalities of
the of the BIAA substrates (phenyl ring, imino-oxy, and carboxy
9
J. AM. CHEM. SOC. VOL. 131, NO. 29, 2009 10315

A R T I C L E S
McIntyre et al.
Table 1. Kinetic Parameters Determined from the Steady-State Preferred Bi-Substrate Michaelis-Menten Eq 3
VMAX(s-1
)
KD,BIAA(mM)
KM, O2(mM)
(VMAX/KM)O2(M-1s-1
)
DKD,BIAA
D(VMAX
)
D(VMAX/KM)O2
protiated
deuterated
4.75 (0.19)
5.68 (0.46)
758.18 (133.42)
367.95 (66.58)
95.37 (12.18)
658.08 (95.02)
4.98 × 1004 (5.03 × 1003)
2.06 (0.52)
0.84 (0.08)
5.78 (0.71)
8.63 × 1003 (6.02 × 1002)
termini moieties) remaining constant with respect to starting
points. At the ‘saddle point’ of the CuII-O• simulation, an
increase in imino-oxy T R-carbon bond length was observed
during a small RC window with a 1.47 Å change between RC
) 1.78 and 2.09 yielding a final imino-oxy T R-carbon bond
of 2.91 Å (Scheme C and movie B, Supporting Information).
equilibrium. The dissociation constant (KD,A[B]) represents the
affinity of the enzyme for the first bound substrate. Analysis
of deuterium KIEs for a steady-state preferred mechanism,
will include the KM for the first bound substrate (KM,A[B]) in
the denominator, yielding a symmetrical mechanism. There-
fore, the D(VMAX/KM)APPARENT value cleavage will be dependent
on the concentration of the second substrate for a steady-state
preferred mechanism, as we observed for CR-D/-H bond
cleavage using BIAA as a substrate. The difference between a
steady-state ordered and a random mechanisms is that the
D(VMAX/KM)APPARENT values approach unity as concentration of
the second substrate becomes infinite for ordered substrate
addition vs a nonunity limit for the random substrate addition.62
Discussion
Dealkylation is Non-enzymatic. Oxidation of BIAA by PAM
yields two products in a 1:1 molar ratio: BOX and glyoxylate.
BOX was confirmed by 13C NMR Figure B, (Supporting
Information). In addition, the stoichiometry of [O2]consumed
:
[BOX]produced was also 1:1 Figure C and Table C (Supporting
Information), consistent with evidence for tight coupling
between substrate oxidation and O2 consumption for PHM61
and the related enzyme, DꢀM.20 PAM-catalyzed oxidation/
dealkylation of BIAA is unaffected by R-hydroxyhippurate (a
PAL substrate) and the PAL-catalyzed dealkylation of R-hy-
droxyhippurate is unaffected by BIAA (Figures 2 and 3). This
suggests that the putative R-hydroxylated product generated by
PHM, C6H5-CH-N-O-CH(OH)-COOH, is unstable and nonen-
zymatically decomposes into observed oxime and glyoxylate
products. These data provide strong evidence that the oxidative
dealkylation of BIAA to BOX and glyoxylate requires only the
PHM domain of bifunctional PAM.
Kinetic Mechanism. Previous work with small-molecule sub-
strates, N-benzoylglycine48 and N-acetylglycine (N. R. McIntyre
and Merkler, D. J., manuscript in preparation), found that
D(VMAX/KM) values for both were equal and constant as the O2
concentration was varied. The minimal kinetic mechanism of these
small-molecule substrates is equilibrium-ordered with O2 binding
after N-benzoylglycine or N-acetylglycine. Comparison of eqs 3
and 4 shows that KM for the first bound substrate (KM,A[B]) is absent
from the denominator for an equilibrium-preferred mechanism. In
this case, binding of the first substrate to the free enzyme (E) is in
The values of D(VMAX/KM)APPARENT,
showed a dramatic
BIAA
decrease as the concentration of O2 increased. Extrapolation of
D
the (VMAX/KM)APPARENT, BIAA to zero [O2] yields a value ∼15
(well beyond the semiclassical limit), while extrapolation of
infinite [O2] yields of 1.0 (Figures C and D, Supporting
Information). The D(VMAX/KM)APPARENT, O2 was constant over the
range [BIAA] used. This is a clear example of a steady-state
ordered mechanism with BIAA binding to reduced PHM prior
to O2 (Scheme 5). Using hippurate as the oxidizable substrate,
it has been established that PHM exhibits an equilibrium-
ordered mechanism with O2 binding after hippurate to reduced
enzyme,48 while the kinetic mechanism for the Y318F mutant
is steady-state random with either hippurate or O2 binding
to reduced enzyme.63 A steady-state random mechanism was
observed for the Y318F mutant of PHM as the a nonunity
values of D(VMAX/KM)APPARENT, HIPPURATE were obtained as [O2]
f ∞.63 For the related enzyme, DꢀM, the absence of the
anionic activator (fumarate) at pH 6.0 alters the steady-state
mechanism from ordered to random.50,64
The steady-state mechanism is attained for BIAA through a
decrease in the rate constant responsible for dissociation of the
binary complex (E-S), k2.48 Unlike an equilibrium preferred
mechanism, the binary complex (E-S) becomes the predomi-
nant enzyme state. The reduction in D(VMAX/KM)APPARENT,BIAA
with increasing [O2] was due to an increase in the “commitment
to catalysis”.65-67 Under steady-state ordered conditions, as [O2]
f ∞, dissociation of the reactant (RCR-H) from the central
complex does not occur as the commitment to the forward
reaction also becomes infinite (D(VMAX/KM)APPARENT,BIAA ) ∼1).
The magnitude of D(VMAX/KM)BIAA as [O2] f 0, has a commit-
ment to the forward reaction which is dependent on the ratio of
the chemical step vs the dissociation of O2 from the central
complex (k5/k4, Scheme 5). Therefore, the predominant enzyme
form would be E-RCR-H due to the decrease in the rate constant
for O2 insertion to achieve the central complex, k3[O2].
(61) Evans, J. P.; Blackburn, N. J.; Klinman, J. P. Biochemistry 2006, 45,
15419–15429.
(62) Cook, P. F.; Cleland, W. W. Biochemistry 1981, 20, 1790–1796.
(63) Francisco, W. A.; Blackburn, N. J.; Klinman, J. P. Biochemistry 2003,
42, 1813–1819.
(64) Miller, S. M.; Klinman, J. P. Biochemistry 1985, 24, 2114–2127.
(65) Northrop, D. B.; Cho, Y. K. Biophys. J. 2000, 79, 1621–1628.
(66) Cleland, W. W. CRC Crit. ReV. Biochem. 1982, 13, 385–428.
(67) Blanchard, J. S.; Cleland, W. W. Biochemistry 1980, 19, 4506–4513.
Figure 4. Dependence of [oxygen] on D(VMAX/KM)APPARENT,
(black
(blue
BIAA
symbols) and the [BIAA] dependence on D(VMAX/KM)APPARENT,
O2
symbols).
9
10316 J. AM. CHEM. SOC. VOL. 131, NO. 29, 2009

Alcohol Intermediate in the Reaction Catalyzed by PHM
A R T I C L E S
Figure 5. QM/MM simulated reaction coordinate for the oxidation chemistry observed for the BIAA CR-radical oxidation with a singlet CuII-OH species
(circles). Squares represent dealkylation distances observed for imino-oxy T CR bond lengths versus reaction coordinate.
steady-state ordered vs equilibrium ordered. The forward
commitment for an equilibrium order mechanism will be
relatively low.
While neither hippurate nor BIAA showed an appreciable
DVMAX, the turnover number decreased approximately 6-fold for
BIAA relative to hippurate. This difference in product release
(VMAX) for BIAA cannot be attributed to an uncoupling of O2
consumption from BOX and glyoxylate formation (Figures 1,
B and Table B), as the reaction stoichiometries for BIAA
oxidation match those reported for glycine-extended substrates.72
One possible reason for the relatively low VMAX for BIAA is
that the imino-oxy moiety is complicating CR-O insertion into
its respective CR-radical species. This conclusion would mean
that BIAA dealkylation would be included in the minimal kinetic
mechanism and would require no other rate constant for product
release other than k7. This rate constant describes the slow step
responsible for the irreversible conversion of the E ·P complex
to free enzyme (E) and product (P) for all PHM substrates.
Figure 6. QM/MM simulated reaction coordinate for the oxidation
chemistry observed for the BIAA CR-radical oxidation with a quartet
CuII-O• species (circles). Squares represent dealkylation distances observed
for imino-oxy T CR bond lengths versus reaction coordinate.
D
Therefore, the absence of VMAX may best be explained as a
requisite rate-limiting conformational change necessary for
product release with glycine-extended substrates, though an
additional energetic penalty associated with BIAA oxidation
(dealkylation) appears to further decrease the magnitude of k7
for this substrate.
The dissociation constant for BIAA showed a normal isotope
D
effect of KD,BIAA ) 2.06 ( 0.52 (Table 1). Conversely, the
binding isotope effect for hippurate is inverse for both PHM
and PAM, KD,hippurate < 1.0.43 Often the effect of isotopic
D
labeling on binding is dismissed as negligible, but binding
isotope effects have been reported for a number of enzymes,
including lactate dehydrogenase,68 hexokinase,69 thymidine
phosphorylase,70 and purine nucleoside phosphorylase.71 The
DKD,BIAA suggests that deuteration of the BIAA R-carbon reduces
stringent vibrational contributions associated with ground-state
destabilization during formation of the E ·BIAA complex
formation, potentially decreasing k2. A decrease in k2 increases
the forward commitment to catalysis, k3/k2, and may be a
contributing factor in the kinetic mechanism for BIAA being
Substrate Oxidation and Product Release are Uncoupled.
QM/MM simulation of both the oxygen insertion into the BIAA
CR-radical and the resulting dealkylation process provided an
experimental constraint used to probe the nature of the Cu/O
oxidant species. The CuII-OH (singlet) species was postulated
to affect the direct OH transfer and product release steps of the
‘side-on/η2’ and ‘copper-oxo pathway B’ mechanisms. The
effect of direct product formation on the bond length of our
dealkylation probe was negligible, suggesting that imino-oxy
T CR bond dissociation would likely not occur through this
mechanism. Formation of the copperII-alkoxide species (quartet)
had a dramatic effect on the structure of the dealkylation probe,
suggesting that the process for substrate oxidation and product
release were completely uncoupled, and likely to pass through
(68) LaReau, R.; Wah, W.; Anderson, V. Biochemistry 1989, 34, 6050–
6058.
(69) Lewis, B. E.; Schramm, V. L. J. Am. Chem. Soc. 2003, 125, 4785–
4798.
(70) Birck, M. R.; Schramm, V. L. J. Am. Chem. Soc. 2004, 126, 6882–
6883.
(71) Murkin, A. S.; Birck, M. R.; Rinaldo-Matthis, A.; Shi, W.; Taylor,
E. A. Biochemistry 2007, 46, 5038–5049.
(72) Kulathila, R.; Merkler, K. A.; Merkler, D. J. Nat. Prod. Rep. 1999,
16, 145–54.
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A R T I C L E S
McIntyre et al.
Scheme 5. Minimal Kinetic Mechanism Determined for BIAA Oxidation As Determined by Primary Deuterium KIEs
Scheme 6. Dealkylation Reaction Predicted for the CuII-Alkoxide Intermediate Formation with BIAA As the Substrate
a copper-alkoxide intermediate (red coordinates, Figure 7). The
doublet copper-oxo species (‘copper-oxo pathway A’) could not
be used for this analysis as H-abstraction and oxidation steps
were concerted resulting in an absence of a substrate intermedi-
ate (radical).37 As the separation of H-abstraction from substrate
oxidation was a necessary caveat for our theoretical treatment
of dealkylation, calculations using the double species could not
be performed.
of the oxime moiety is electronically analogous to an amide
(manuscript in preparation). For the QM/MM study, CR-H
bond cleavage was not part of the reaction coordinate; instead
the dealkylation of BIAA was used to provide the system with
an independent variable to constrain the theoretical model. The
mixture of both in Vitro and in silico data suggests that direct
hydroxyl insertion into the CR-radical of BIAA is unlikely as
the NO T CR distance did not significantly increase. Interest-
ingly, the singlet species reaction coordinate was thermody-
namically favored as the oxidant with high symmetry over the
simulation. This mechanism would likely lead to a complete
uncoupling of the BIAA radical and a reduced oxygen species
from the PHM active site, as the substrate radical would resist
conversion into a thermodynamically unstable hemiacetal
derivative. PHM would not be expected to stabilize the putative
HO-CR BIAA intermediate as the active site is highly solvated
during catalysis.73 Thus, if this hemiacetal formed, its instability
would not be suppressed through protein docking.
Using the natural glycine-extended substrate, hydrolysis of
the ‘copper-alkoxide/substrate’ intermediate complex yields a
stable R-hydroxylated glycine product. The BIAA model
displayed changes in the bond lengths for the oxygen within
the CuIITO• complex approaching the carbon radical (a
decrease) as NOTCR (an increase) over the reaction coordinate
(Figure 7). The steady-state data suggest that this process is
irreversible and tightly coupled with respect to both oxygen and
BIAA, similar to the PHM natural substrate.20 Taken together,
the reason for the O-dealkylation of BIAA occurring indepen-
dently of PAL is likely that the oxidation step becomes
increasingly rate limiting versus the subsequent hydrolysis step
in BIAA compared to a glycine-extended substrate. This
irreversible event was calculated to be an endothermic process
(Figure 7) in both singlet and quartet copper-oxo complexes,
though O-dealkylation of BIAA was only observed for the
singlet-state species calculation. By comparison, for glycine-
extended substrates, this event has been calculated to be an
exothermic process.19,35
Conclusion
Due to the complexities of PHM, theoretical models lacking
experimental constraint often lead to ambiguous conclusions
in thermodynamic preference between chemical models. Here,
the catalytic power of the enzyme is determined at its upper
limit and does not take into account that the reaction regime
only requires the necessary energy for catalysis. The work of
Karlin (supra Vide) infers that the nature of the Cu/O oxidant
cannot definitively characterized in PHM, as the reaction
coordinate may proceed through several possible catalytic paths.
Therefore, the dealkylation event was designed to describe the
nature of substrate oxidation chemistry in PHM. BIAA was a
superb probe for the glycine-extended substrate functionalization
as it retained all reaction stoichiometries important to PHM.
When docked to PHM, non-Lewis hyperconjugative stabilization
Our proposal that the ‘CuII-O•’ species was the oxidant in
PHM is based on the dealkylation characteristics unique to
BIAA. Formation of the ‘inner-sphere alcohol’ complex during
BIAA oxidation was the only mechanism which could account
for the tightly coupled reaction stoichiometries while still
displaying the ability to dealkylate independently of PAL. The
copperII-alkoxide was thermodynamically unfavored compared
Figure 7. Combined plot of QM/MM reaction coordinate simulation
comparing the CuII-OH (singlet) and the CuII-O• (quartet) species against
bond distances for the BIAA dealkylation event. Please note, squares
represent the distance change between the NOTCR bond distance, while
the circles signify CufO bond distance changes over the reaction
coordinate. For more details, please see the Materials and Methods sec-
tion.
(73) Owen, T. C.; Merkler, D. J. Med. Hypotheses 2004, 62, 392–400.
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Alcohol Intermediate in the Reaction Catalyzed by PHM
A R T I C L E S
to the singlet CuII-OH species, though it was the increase in
NO T CR bond length that pointed strongly to an ‘inner-sphere
alcohol’ intermediate. Therefore, production of an oxidized
copperII-alkoxide intermediate complex would be favored to
facilitate the reaction characteristics observed during the BIAA
O-dealkylation process (Scheme 6). In conclusion, this study
provides compelling evidence that PHM-dependent oxidation
of glycine-extended substrates occurs through a covalently
linked copperII-alkoxide substrate complex.
Research Foundation, the Milheim Foundation for Cancer Research,
the Shin Foundation for Medical Research, the Wendy Will Case
Cancer Fund, and the University of South Florida - Established
Researcher Grant Program, to D.J.M, and a predoctoral fellowship
to E.W.L. from the American Heart Association (0415259B). The
authors would like to acknowledge the use of the services provided
by Research Computing, University of South Florida. This research
was also supported by an allocation through the TeraGrid Advanced
Support Program (TG-MCB070112N).
Acknowledgment. This work was supported, in part, by grants
from the National Institutes of Health - General Medical Sciences
(R15-GM067257 and R15-GM073659), the Shirley W. and William
L. Griffin Foundation, the Alpha Research Foundation, Inc., the
Eppley Foundation for Research, the Gustavus and Louise Pfieffer
Supporting Information Available: Experimental figures,
tables, schemes, and movies. This material is available free of
charge via the Internet at http://pubs.acs.org.
JA902716D
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