Evidence for substrate preorganization in the peptidylglycine α-amidating monooxygenase reaction describing the contribution of ground state structure to hydrogen tunneling

Article abstract of DOI:10.1021/ja1019194

Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme which catalyzes the post-translational modification of inactive C-terminal glycine-extended peptide precursors to the corresponding bioactive α-amidated peptide hormone. This conversion involves two sequential reactions both of which are catalyzed by the separate catalytic domains of PAM. The first step, the copper-, ascorbate-, and O₂-dependent stereospecific hydroxylation at the α-carbon of the C-terminal glycine, is catalyzed by peptidylglycine α-hydroxylating monooxygenase (PHM). The second step, the zinc-dependent dealkylation of the carbinolamide intermediate, is catalyzed by peptidylglycine amidoglycolate lyase. Quantum mechanical tunneling dominates PHM-dependent C_α-H bond activation. This study probes the substrate structure dependence of this chemistry using a set of N-acylglycine substrates of varying hydrophobicity. Primary deuterium kinetic isotope effects (KIEs), molecular mechanical docking, alchemical free energy perturbation, and equilibrium molecular dynamics were used to study the role played by ground-state substrate structure on PHM catalysis. Our data show that all N-acylglycines bind sequentially to PHM in an equilibrium-ordered fashion. The primary deuterium KIE displays a linear decrease with respect to acyl chain length for straight-chain N-acylglycine substrates. Docking orientation of these substrates displayed increased dissociation energy proportional to hydrophobic pocket interaction. The decrease in KIE with hydrophobicity was attributed to a preorganization event which decreased reorganization energy by decreasing the conformational sampling associated with ground state substrate binding. This is the first example of preorganization in the family of noncoupled copper monooxygenases.

Full text of DOI:10.1021/ja1019194

Published on Web 11/02/2010
Evidence for Substrate Preorganization in the Peptidylglycine
r-Amidating Monooxygenase Reaction Describing the
Contribution of Ground State Structure to Hydrogen Tunneling
Neil R. McIntyre,† Edward W. Lowe, Jr.,‡,§ Jonathan L. Belof,‡ Milena Ivkovic,‡
Jacob Shafer,‡ Brian Space,‡ and David J. Merkler*,‡
Department of Chemistry, XaVier UniVersity of Louisiana, 1 Drexel DriVe, New Orleans,
Louisiana 70125, United States, and Department of Chemistry, UniVersity of South Florida,
4202 East Fowler AVenue, Tampa, Florida 33620, United States
Received March 8, 2010; E-mail: merkler@usf.edu
Abstract: Peptidylglycine R-amidating monooxygenase (PAM) is a bifunctional enzyme which catalyzes
the post-translational modification of inactive C-terminal glycine-extended peptide precursors to the
corresponding bioactive R-amidated peptide hormone. This conversion involves two sequential reactions
both of which are catalyzed by the separate catalytic domains of PAM. The first step, the copper-, ascorbate-,
and O2-dependent stereospecific hydroxylation at the R-carbon of the C-terminal glycine, is catalyzed by
peptidylglycine R-hydroxylating monooxygenase (PHM). The second step, the zinc-dependent dealkylation
of the carbinolamide intermediate, is catalyzed by peptidylglycine amidoglycolate lyase. Quantum mechanical
tunneling dominates PHM-dependent CR-H bond activation. This study probes the substrate structure
dependence of this chemistry using a set of N-acylglycine substrates of varying hydrophobicity. Primary
deuterium kinetic isotope effects (KIEs), molecular mechanical docking, alchemical free energy perturbation,
and equilibrium molecular dynamics were used to study the role played by ground-state substrate structure
on PHM catalysis. Our data show that all Ν-acylglycines bind sequentially to PHM in an equilibrium-ordered
fashion. The primary deuterium KIE displays a linear decrease with respect to acyl chain length for straight-
chain N-acylglycine substrates. Docking orientation of these substrates displayed increased dissociation
energy proportional to hydrophobic pocket interaction. The decrease in KIE with hydrophobicity was attributed
to a preorganization event which decreased reorganization energy by decreasing the conformational
sampling associated with ground state substrate binding. This is the first example of preorganization in the
family of noncoupled copper monooxygenases.
hormones in mammals,1a insects,4 and cnidarians5 and also may
have a role in the biosynthesis of mammalian lipid amides such
Introduction
Peptidylglycine R-amidating monooxygenase (PAM) is a
bifunctional metallo-oxygenase of considerable interest across
many fields, including neurobiology, mechanistic enzymology,
metabolomics, and process biochemistry.1 Industrially, PAM has
been used for the in vitro production of salmon calcitonin.2 In
vivo, PAM catalyzes the oxidative cleavage of the glycyl CR-N
bond yielding an amide and glyoxylate (Scheme 1).3 This
chemistry is essential to biosynthesis of R-amidated peptide
as oleamide.6
The amidation reaction, as catalyzed by PAM, occurs in two
steps.7 The first step is the copper-dependent hydroxylation of
the glycyl R-carbon in a reaction that requires O2 and a
reductant.3b,7b,8 The second step is the dealkylation of carbinol-
amide in a reaction that requires zinc.9 Metals other than zinc
have been reported to be important for the dealkylation
(4) (a) Vanden Broeck, J. Peptides 2001, 22, 241–254. (b) Predel, R. In
InVertebrate Neuropeptides and Hormones: Basic Knowledge and
Recent AdVances 2006; Satake, H., Ed.; Transworld Research Network:
Kerala, India, 2006; pp 127-155.
† Xavier University of Louisiana.
‡ University of South Florida.
§ Present address: Vanderbilt University Center for Structural Biology,
465 21st Ave. South BIOSCI/MRBIII, Room 5144F, Nashville, TN 37232-
8725.
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McIntyre et al.
Scheme 1. Reactions Catalyzed by Bifunctional PAMa
a Peptidylglycine R-hydroxylating monooxygenase (PHM) domain is dependent on copper reduction by two ascorbate (ASC) molecules to activate the
Cu/O2 species for CR-Hs cleavage and hydroxylation. Deakylation of the stable R-hydroxylated intermediate is catalyzed by the peptidylamidoglycolate lyase
(PAL) domain to yield glyoxylate and the corresponding amide. Oxidation of ascorbate yields semidehydroascorbate (SDA).
reaction,10 but the most recent data suggest a direct catalytic
role only for zinc.11
likely ascorbic acid in vivo. Reduction is ping-pong with the
release of oxidized reductant from E ·2Cu(I) followed by binding
O2 or oxidizable substrate to the reduced enzyme.18,19 The
dependence of the 18O-kinetic isotope effect on deuteration of
the oxidizable substrate for both enzymes indicates that C-H
bond cleavage precedes cleavage of the activated O2 complex,
yielding a Cu(II)-hydroperoxo and a substrate-based radical.20
The nucleophilic species responsible for hydrogen abstraction
is a Cu(II)-superoxo.21 The oxidative species responsible for
hydroxylation of the aliphatic radical is the subject of much
debate.1e,22 Recent data from our laboratory suggest that an end-
on/η1 Cu(II)-oxyl is the oxidant in PHM.19
Klinman and co-workers have demonstrated that CR-H bond
cleavage as catalyzed by PHM is dominated by quantum mechan-
ical tunneling.20b,23 This result was surprising because of the high
degree of solvent accessibility of the PHM active site.13 Detailed
studies of enzymes catalyzing H-transfer reactions have demon-
strated that semiclassical transition state theory, including the Bell
tunneling correction, cannot adequately account for the wealth of
the kinetic isotope effect obtained on these systems.24-26 Over the
past few years, a full tunneling model has emerged that can account
Peptidylglycine R-hydroxylating monooxygenase (PHM)
shares mechanistic, sequence, and structural homology with
dopamine ꢀ-monooxygenase (DꢀM).1e Both enzymes contain
two bound copper atoms that are required for catalytic activity.8,12
The crystal structure of PHM shows that the two copper atoms
are separated by a distance of 10.6 Å across a solvent-filled
active site,13 results consistent with extended X-ray absorption
fine structure (EXAFS) data for DꢀM indicating that two DꢀM-
bound copper atoms were >4 Å apart.14 Electron paramagnetic
resonance (EPR) and EXAFS studies of PHM and DꢀM indicate
that the active site copper atoms behave completely as nonblue
type II, uncoupled mononuclear metal centers.15 Consistent with
the EPR and EXAFS data is the finding that the two PHM-
bound copper atoms have different functions in catalysis and
amino acid ligands.15,16 One copper atom, CuH, has three Nδ-
histidine ligands (His107, His108, and His172) and is involved in
electron transfer. The other copper atom, CuM, has two Nε-
histidines ligands (His240 and His242) and a methionine sulfur
ligand (Met314) and is involved in O2 activation and substrate
oxidation.13
The reactions catalyzed by PHM and DꢀM involve hydrogen
atom transfer from substrate to an activated Cu/O species,
resulting either in the stereospecific hydroxylation of a glycyl
R-carbon (PHM) or benzylic carbon (DꢀM).7a,17 The first step
in the catalytic cycle for both enzymes is reduction of the
enzyme-bound Cu(II) atoms with an exogenous reductant, most
(17) (a) Ramer, S. E.; Cheng, H.; Palcic, M. M.; Vederas, J. C. J. Am.
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A R T I C L E S
for the kinetic isotope effect data obtained for H-transfer enzymes,
including their pressure-dependence.24-27 The hallmark of this
model is the coupling of environmental reorganization to
enzyme-catalyzed hydrogen tunneling (for the transfer of H+,
H-, or H·) through the reaction barrier. Environmental reor-
ganization involves two types of motions which are coupled to
H-tunneling, (a) Marcus-like, thermally equilibrated, relatively
slow (ns - ms) motions of the enzyme and substrate which
increase the possibility of attaining an E ·S configuration
optimized for tunneling (referred to as preorganization) and (b)
nonequilibrium, relatively rapid (fs - ps) “gating” motions that
optimize the H-donor and acceptor distance for wave function
overlap (referred to as reorganization or protein promoting
vibrations). The parameters most important in relating environ-
mental reorganization to tunneling probability is the reorganiza-
tion energy, λ, and the gating frequency, ωg.24,25 Work on
soybean lipoxygenase (SLO) and PHM has shown that the
magnitude of the λ is approximately the same for both, 19.5
kcal/mol for SLO25 and 20 kcal/mol for PHM.23 However, the
gating frequencies are different for the two enzymes, 400 cm-1
for SLO and 45 cm-1 for PHM.23 This difference in ωg between
the two enzymes likely results from the increased flexibility of
PHM relative to more rigid SLO with a greater participation of
the gating motions in PHM catalysis. The SLO active site is
nonpolar to accommodate the binding of fatty acid substrates
rendering the gating motions energetically difficult. Thus, H ·
tunneling in SLO is dominated by active site preorganization
with only a small contribution from gating motions.
as an indirect probe into the essential role protein dynamics
contributes to the reaction coordinate.
The present study deepens our understanding of the underly-
ing relationship between PHM dynamics and substrate structure
and is built upon the earlier work of Wilcox et al.29 and Merkler
et al.30 Herein, we define how substrate hydrophobicity affects
equilibrium binding to reduced PHM in the ground state and
CR-H cleavage, as observed in the steady-state. Using a series
of unbranched, nonconjugated, N-acyglycines (from N-acetyl-
glycine to N-decanoylglycine), we determined the acyl chain-
length effect on both the primary kinetic isotope effects (KIEs)
for CR-H bond cleavage and the viscosity-dependence of
apparent VMAX and VMAX/KM terms. Alchemical free energy
perturbation calculations (AFEP) and equilibrium molecular
dynamics simulations were performed in conjunction with the
biochemical experiments to define the relationship between acyl
chain length of N-acylglycine substrates and the relative
dissociation energies of the reduced PHM ·acylglycine and the
reduced PHM ·acylglycine·O2 complexes. Overall, our data
suggest that N-acylglycine binding utilizes hydrophobic interac-
tions coupled to salt-bridge formation with Arg240 in the
enzyme·acylglycine·O2 central complex as the likely mecha-
nism by which PHM preorganization regulates catalysis. Our
work provides the first evidence of preorganization in the family
of noncoupled dicopper monooxygenases and yields a novel
perspective on the role the ground state substrate structure has
on conformational dynamics in the PHM tunneling reaction.
Francisco et al.23 provided the first evidence of tunneling in
the hydrogen atom abstraction reaction catalyzed by PHM. The
hallmark features of this study, using N-benzoylglycine as a
substrate, were a temperature-independent intrinsic primary
deuterium KIE of 10.6 ( 0.8, an Arrhenius prefactor deuterium
isotope effect of 5.9 ( 3.2, and an Ea(D) - Ea(H) value of 0.37
( 0.33 kcal/mol. All of these experimentally determined values
are well outside the semiclassical limits for C-H bond cleavage
pointing toward a tunneling mechanism. Fits of these data to
the model incorporating environmentally coupled motions
pointed toward an important participation of gating motions in
PHM catalysis. This result seems consistent with the large,
solvent-accessible PHM active site (required to accommodate
peptide substrates that can be in excess of 100 amino acids long)
that is not likely optimized for tunneling in the ground state.
Evidence that more global conformational dynamics are also
important in PHM catalysis comes from pH-dependent changes
in the Debye-Waller factor for the Cu-SMet314 component of
the X-ray absorption spectrum of reduced enzyme.28 Bauman
et al.28 report that reduced PHM exists in two forms: an inactive
“met-on” form with a strong Cu-SMet314 interaction and an
active “met-off” form with a relative weak Cu-SMet314 interac-
tion. These authors found that the pH dependence of “met-off”
f “met-on” transition and the PHM reaction was similar (pKa
∼ 5.9). The transition between reduced Cu-SMet314 substates
resulted from a global change in PHM structure with confor-
mational mobility associated with the “met-off” state serving
Materials and Methods
Materials. Morpholino-ethane-sulfonic acid (MES), sodium
ascorbate, acetyl chloride, propionic anhydride, butyric anhydride,
hexanoic anhydride, octanoic anhydride, decanoic anhydride, and
cuprous nitrate were obtained from Sigma, N-acetylglycine was
purchased from TCI, America, [R-2H2]-glycine (98%) was pur-
chased from CDN Isotopes, Triton-X-100 was purchased from
Fisher, and bovine liver catalase was supplied by Worthington.
Recombinant type A rat medullary thyroid carcinoma bifunctional
PAM was produced and purified as described31 and was a gift from
Unigene Laboratories, Inc. (Fairfield, NJ, see www.unigene.com).
All other experimental reagents were purchased from commercial
sources at the highest purity grade available and used without
additional modification.
N-Acylglycine and [r-2H2]-N-Acylglycine Synthesis. The N-
acylglycines and the [R-2H2]-N-acylglycines were synthesized
according to literature procedures.29,32 To a cooled solution of
glycine or [R-2H2]-glycine at 0 °C containing 1.2 equiv of NaOH,
1 equiv of the desired acyl anhydride was added dropwise with
stirring. The reaction was allowed to stir for an additional 3 h with
the temperature slowly rising to room temperature and the pH
maintained at 7-8 by the manual addition of NaOH, as necessary.
An acid extraction of the aqueous phase into EtOAc (3×) was
washed with brine and was subsequently dried over anhydrous
MgSO4. Solvent was removed in vacuo, the product first recrystal-
lized from a minimum volume of hot ethyl acetate, and then
precipitated with hexane to yield white crystals. The NMR spectra
(1H and 13C) for the N-acylglycines we synthesized are provided
in the Supporting Information.
(25) Knapp, M. J.; Rickert, K.; Klinman, J. P. J. Am. Chem. Soc. 2002,
124, 3865–3874.
(29) Wilcox, B. J.; et al. Biochemistry 1999, 38, 3235–3245.
(30) Merkler, D. J.; et al. Bioorg. Med. Chem. 2008, 16, 10061–10074.
(31) Miller, D. A.; Sayad, K. U.; Kulathila, R.; Beaudry, G. A.; Merkler,
D. J.; Bertelsen, A. H. Arch. Biochem. Biophys. 1992, 298, 380–388.
(32) (a) Mesaik, M. A.; Rahat, S.; Khan, K. M.; Ullah, Z.; Choudhary,
M. I.; Murad, S.; Ismail, Z.; Rahman, A.; Ahmad, A. Bioorg. Med.
Chem. 2004, 12, 2049–2057. (b) Katz, J.; Lieberman, I.; Barker, H. A.
J. Biol. Chem. 1953, 200, 431–441. (c) Jones, W. D. J. Chem. Soc.
Perkin I 1981, 344–348.
(26) (a) Sutcliffe, M. J.; Scrutton, N. S. Phys. Chem. Chem. Phys. 2006, 8,
4510–4516. (b) Hay, S.; Pudney, C.; Hothi, P.; Johannissen, L. O.;
Masgrau, L.; Pang, J.; Leys, D.; Sutcliffe, M. J.; Scrutton, N. S.
Biochem. Soc. Trans. 2008, 36, 16–21.
(27) Hay, S.; Scrutton, N. S. Biochemistry 2008, 47, 9880–9887.
(28) Bauman, A. T.; Jaron, S.; Yul, E. T.; Burchfiel, J. R.; Blackburn, N. J.
Biochemistry 2006, 45, 11140–11150.
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McIntyre et al.
[2H2]-N-Acetylglycine. 1H NMR (400 MHz, Me2SO-d6) δ 1.80
(singlet, 3H, CH3) δ 8.10(singlet, 1H, NH). 13C NMR (100 MHz,
Me2SO-d6) δ 172.1 (CdO, carboxylic acid), δ170. Three (CdO,
amide), δ 22.9 (CH3, methyl) mp. 205-207 °C (lit. 208 °C).32a
(CH2, n-alkyl methylene linker), δ 14.6 (CH3, n-alkyl terminal
methyl). mp. 102-104 °C.
1
N-Decanoylglycine. H NMR (400 MHz, Me2SO-d6) δ 0.84
(triplet, J ) 6.6 Hz, 3H, CH3), δ 1.24 (multiplet, 12H, (CH2)6), δ
1.48 (multiplet, 2H, CH2), δ 2.08 (triplet, J ) 7.4 Hz, 2H, CH2), δ
3.70 (singlet, 2H), and δ 8.08 (singlet, 1H). 13C NMR (100 MHz,
Me2SO-d6) δ 173.2 (CdO, carboxylic acid), δ 172.1 (CdO, amide),
δ 41.7 (CH2, R-glycine), δ 35.7 (CH2, n-alkyl methylene linker), δ
31.9 (CH2, n-alkyl methylene linker), δ 29.5 ((CH2)3, n-alkyl
methylene linker), δ 25.8 ((CH2)2, n-alkyl methylene linker), δ 22.7
(CH2, n-alkyl methylene linker), δ 14.5 (CH3, n-alkyl terminal
methyl). mp. 113-114 °C (lit. 114 °C).32c
1
N-Propionylglycine. H NMR (400 MHz, Me2SO-d6) δ 0.91
(triplet, J ) 7.6 Hz, 3H, CH3), δ 2.07 (quartet, J ) 7.6 Hz, 2H,
CH2), δ 3.67 (singlet, 2H, CH2), and δ 7.96 (singlet, 1H, NH). 13
C
NMR (100 MHz, Me2SO-d6) δ 174.5 (CdO, carboxylic acid), δ
172.1 (CdO, amide), δ 41.2 (CH2, R-glycine), δ 28.8 (CH2, n-alkyl
methylene linker), δ 10.2 (CH3, n-alkyl terminal methyl). mp.
122-124 °C (lit. 125.5-7 °C).32b
1
[2H2]-N-Propionylglycine. H NMR (400 MHz, Me2SO-d6) δ
1
[2H2]-N-Decanoylglycine. H NMR (400 MHz, Me2SO-d6) δ
0.93 (triplet, J ) 7.6 Hz, 3H, CH3), δ 2.07 (quartet, J ) 7.2 Hz,
2H, CH2), and δ 8.01 (singlet, 1H, NH). 13C NMR (100 MHz,
Me2SO-d6) δ 173.9 (CdO, carboxylic acid), δ 172.1 (CdO, amide),
δ 28.8 (CH2, n-alkyl methylene linker), δ 10.4 (CH3, n-alkyl
terminal methyl). mp. 122-124 °C.
0.67 (triplet, J ) 6.0 Hz, 3H, CH3), δ 1.06 (multiplet, 12H, (CH2)6),
δ 1.31 (multiplet, 2H, CH2), δ 1.92 (triplet, J ) 7.2 Hz, 2H, CH2),
and δ 7.89 (singlet, 1H). 13C NMR (100 MHz, Me2SO-d6) δ 173.3
(CdO, carboxylic acid), δ 172.1 (CdO, amide), δ 35.7 (CH2,
n-alkyl methylene linker), δ 31.0 (CH2, n-alkyl methylene linker),
δ 30.0 ((CH2)3, n-alkyl methylene linker), δ 25.8 ((CH2)2, n-alkyl
methylene linker), δ 22.8 (CH2, n-alkyl methylene linker), δ 14.5
(CH3, n-alkyl terminal methyl). mp. 113-114 °C.
N-Butyrylglycine. 1H NMR (400 MHz, Me2SO-d6) δ 0.80
(triplet, J ) 7.4 Hz, 3H, CH3), δ 1.45 (multiplet, 2H, CH2), δ 2.04
(triplet, J ) 7.2 Hz, 2H, CH2), δ 3.68 (singlet, 2H, CH2), and δ
8.05 (singlet, 1H, NH). 13C NMR (100 MHz, Me2SO-d6) δ 172.5
(CdO, carboxylic acid), δ 171.5 (CdO, amide), δ 40.5 (CH2,
R-glycine), δ 37.0 (CH2, n-alkyl methylene linker), δ 18.6 (CH2,
n-alkyl methylene linker), δ 13.5 (CH3, n-alkyl terminal methyl).
mp. 69-70 °C (lit. 68.5-70 °C).32b
Steady-State Kinetics. Reactions at 37.0 ( 0.1 °C were initiated
by the addition of 0.12-0.18 µM PAM (4-5 µL) into 2.0 mL of
100 mM MES/NaOH pH 6.0, 30 mM NaCl, 1.0% (v/v) ethanol,
0.001% (v/v) Triton X-100, 1.0 µM Cu(NO3)2, 5.0 mM sodium
ascorbate, with N-acylglycine or [R-2H2]-N-acylglycine at concen-
trations of 0.2-10-fold KM. The concentration of dissolved O2 under
these conditions was 217 µM.33 Initial rates were measured by
following the PAM-dependent consumption of O2 using a Yellow
Springs Instrument model 53 oxygen monitor interfaced with a
personal computer using a Dataq Instruments analogue/digital
converter (model DI-154RS). VMAX,app values were normalized to
controls performed at 11.0 mM N-acetylglycine to account for
differences in specific activity between different lots of enzyme.
Background O2 consumption rates were first determined without
enzyme and were subtracted from the rate obtained upon PAM
addition. Ethanol was added to protect the catalase against
ascorbate-mediated inactivation,34 and Triton X-100 was included
to prevent nonspecific absorption of PAM to the sides of the oxygen
monitor chambers.
O2-Dependence of the Primary Deuterium Kinetic Isotope
Effects. The O2-dependence of the KIEs expressed by N-acetyl-
glycine was determined by the addition of 0.18 µM PAM into 2.0
mL of 100 mM MES/NaOH pH 6.0, 30 mM NaCl, 1.0% (v/v)
ethanol, 0.001% (v/v) Triton X-100, 1.0 µM Cu(NO3)2, 5.0 mM
sodium ascorbate, 2-50 mM N-acetylglycine (or [R-2H2]-N-
acetylglycine) and 25-830 µM O2. The [O2] was varied by mixing
different proportions of N2:O2 gas into the headspace of the
electrode chamber above the stirring reaction for 4 min. The
resulting [O2] was determined from percent saturation observed with
the O2 electrode compared to the ambient [O2] as a reference.
Viscosity Dependence of the PAM-Catalyzed Oxidation of
N-Acylglycines. An Ubbelholde viscometer (Industrial Research
Glassware Ltd., Union, NJ, size 1B) was used to determine the
relative microviscosity (ηrel) of the control solution containing 100
mM MES/NaOH pH 6.0, 30 mM NaCl, 1.0% (v/v) ethanol, and
0.001% (v/v) Triton X-100. Ten trials were performed in a
temperature-controlled water bath, with viscometer and buffer
equilibrated for 10 min at 37.0 ( 0.1 °C and the values (centistokes)
averaged with the corresponding standard deviation. The relative
microviscosity was determined by comparing buffer solution
supplemented with sucrose (at the desired concentrations) to the
control solution. The relative microviscosities used were 1.0 (no
microviscogen), 2.08, 3.69, and 5.33. Macroviscosity was measured
in a similar fashion using a Ficoll-400 solution (pH 6.0) to alter
[2H2]-N-Butyrylglycine. 1H NMR (400 MHz, Me2SO-d6) δ 0.72
(triplet, J ) 7.4 Hz, 3H, CH3), δ 1.38 (multiplet, 2H, CH2), δ 1.95
(triplet, J ) 7.2 Hz, 2H, CH2), and δ 7.95 (singlet, 1H, NH). 13C
NMR (100 MHz, Me2SO-d6) δ 172.6 (CdO, carboxylic acid), δ
171.6 (CdO, amide), δ 37.1 (CH2, n-alkyl methylene linker), δ
18.7 (CH2, n-alkyl methylene linker), δ 13.7 (CH3, n-alkyl terminal
methyl). mp. 69-71 °C.
1
N-Hexanoylglycine. H NMR (400 MHz, Me2SO-d6) δ 0.79
(triplet, J ) 7.6 Hz, 3H, CH3), δ 1.24 (multiplet, 4H, (CH2)2), δ
1.51 (multiplet, 2H, CH2), δ 2.09 (triplet, J ) 6.8 Hz, 2H, CH2), δ
3.74 (singlet, 2H, CH2), and δ 8.05 (singlet, 1H, NH). 13C NMR
(100 MHz, Me2SO-d6) δ 173.3 (CdO, carboxylic acid), δ 172.1
(CdO, amide), δ 41.2 (CH2, R-glycine), δ 39.7 (CH2, n-alkyl
methylene linker), δ 35.7 (CH2, n-alkyl methylene linker), δ 25.6
(CH2, n-alkyl methylene linker), δ 22.6 (CH2, n-alkyl methylene
linker), δ 14.6 (CH3, n-alkyl terminal methyl). mp. 88-89 °C (lit.
92 °C).32b
1
[2H2]-N-Hexanoylglycine. H NMR (400 MHz, Me2SO-d6) δ
0.77 (triplet, J ) 6.7 Hz, 3H, CH3), δ 1.17 (multiplet, 4H, (CH2)2),
δ 1.41 (multiplet, 2H, CH2), δ 2.02 (triplet, J ) 7.5 Hz, 2H, CH2),
and δ 7.99 (singlet, 1H, NH). 13C NMR (100 MHz, Me2SO-d6) δ
172.7 (CdO, carboxylic acid), δ 171.5 (CdO, amide), δ 35.1 (CH2,
n-alkyl methylene linker), δ 30.9 (CH2, n-alkyl methylene linker),
δ 24.9 (CH2, n-alkyl methylene linker), δ 21.9 (CH2, n-alkyl
methylene linker), δ 13.9 (CH3, n-alkyl terminal methyl). mp.
88-89 °C.
1
N-Octanoylglycine. H NMR (400 MHz, Me2SO-d6) δ 0.73
(triplet, J ) 6.4 Hz, 3H, CH3), δ 1.14 (multiplet, 8H, (CH2)4), δ
1.38 (multiplet, 2H, CH2), δ 1.98 (triplet, J ) 7.2 Hz, 2H, CH2), δ
3.60 (singlet, 2H, CH2), and δ 7.98 (singlet, 1H, NH). 13C NMR
(100 MHz, Me2SO-d6) δ 173.3 (CdO, carboxylic acid), δ 172.1
(CdO, amide), δ 41.2 (CH2, R-glycine), δ 39.7 (CH2, n-alkyl
methylene linker), δ 35.8 (CH2, n-alkyl methylene linker), δ 31.9
(CH2, n-alkyl methylene linker), δ 29.3 ((CH2)2, n-alkyl methylene
linker), δ 22.8 (CH2, n-alkyl methylene linker), δ 14.6 (CH3, n-alkyl
terminal methyl). mp. 103-105 °C. (lit. 105 °C).32c
1
[2H2]-N-Octanoylglycine. H NMR (400 MHz, Me2SO-d6) δ
0.79 (triplet, J ) 6.4 Hz, 3H, CH3), δ 1.20 (multiplet, 8H, (CH2)4),
δ 1.47 (multiplet, 2H, CH2), δ 2.07 (triplet, J ) 7.2 Hz, 2H, CH2),
and δ 8.01 (singlet, 1H, NH). 13C NMR (100 MHz, Me2SO-d6) δ
173.0 (CdO, carboxylic acid), δ 171.7 (CdO, amide), δ39.9 (CH2,
n-alkyl methylene linker), δ 35.4 (CH2, n-alkyl methylene linker),
δ 31.5 (CH2, n-alkyl methylene linker), δ 28.9 ((CH2)2, n-alkyl
methylene linker), δ 25.5 (CH2, n-alkyl methylene linker), δ 22.4
(33) Morrison, T. J.; Billet, F. J. Chem. Soc. 1952, 3819–3822.
(34) Davison, A. J.; Kettle, A. J.; Fatur, D. J. J. Biol. Chem. 1986, 261,
1193–1200.
9
16396 J. AM. CHEM. SOC. VOL. 132, NO. 46, 2010

A R T I C L E S
the macroviscosity of the reaction environment. The relative
macroviscosities were 1.0 (no macroviscogen), 3.27, and 5.17. The
dependence of the steady-state kinetic parameters was determined
by measuring the consumption of O2 for N-acetylglycine or
N-decanoylglycine in solutions fixed at each relative microviscosity
and macroviscosity. Reactions were initiated by the addition of 0.12
µM PAM into 100 mM MES/NaOH pH 6.0, 30 mM NaCl, 1.0%
(v/v) ethanol, 0.001% (v/v) Triton X-100, 1.0 µM Cu(NO3)2, 5.0
mM sodium ascorbate, N-acetylglycine, or N-decanoylglycine, and
the desired concentration of viscogen (sucrose or Ficoll-type 400).
The VMAX,app values were normalized to 11.0 mM N-acetylglycine
(no viscogen).
Analysis of Steady-State Kinetic Data. Steady-state kinetic
parameters ((standard error) were obtained by a Kaleida-Graph
fit of the initial velocity (rate) vs initial substrate concentration ([S])
to the Michaelis-Menten equation (eq 1) where KM,app is the
apparent Michaelis constant for the N-acylglycine at fixed [ascor-
bate] and [O2] and VMAX,app is the apparent maximum velocity at
saturating N-acylglycine and fixed [ascorbate] and [O2].
Figure 1. The active-site analogue that was used in the electronic structure
calculations. The histidine and methionine residues were methyl-capped
with the methyl cap carbons frozen. This geometry was optimized and then
used in the normal-mode analysis and ESP calculations.
Vmax,app[S]
for reduced PHM precatalytic complex that consists of residues
43-356 of rat PAM that includes the PHM catalytic core. MD
was performed with NAMD 2.6 using the CHARMM22 force field
parameters where available, including treatments for (noncatalytic
site) PHM, N-acylglycines, and N-benzoylglycine (hippurate).39
Additional required parameters for the reactive center were
calculated using the GAMESS electronic structure code on a
representative molecular fragment shown in Figure 1. Density
functional theory40 calculations were performed with the B3LYP41
hybrid exchange-correlation functional and the SBKJC42 basis set
using effective core potentials on the copper. The intramolecular
force field parameters for both CuH and CuM were determined by
normal-mode analysis. Specifically, the force constant matrix was
calculated and diagonalized for each Cu-containing fragment
separately. Partial charges for a subset of the reactive center atoms
in Figure 1 (i.e., copper and the oxygen atoms of O2) were fit to
the electrostatic potential surface using the Connelly algorithm
implemented in GAMESS. The van der Waals parameters for the
Cu and O2 atoms are taken from the UFF43 force field.
rate )
(1)
KM,app + [S]
Values for the D(VMAX/KM)app and DVMAX,app were obtained from
the quotient of appropriate constants for the light/heavy N-
acylglycine substrate.35 Initial rate data generated to determine the
minimal kinetic mechanism were fit to either a steady-state (eq 2)
or equilibrium preferred (eq 3) minimal kinetic mechanism using
the ENZKIN programs, where [AG] is the concentration of the
N-acetylglycine, [O2] is the concentration of O2, VMAX represents
the maximal velocity for the reaction at infinite concentrations of
both O2 and N-acetylglycine, KM values for either substrate are the
Michaelis constant at saturating concentrations of the second
substrate, and KI,AG is the steady-state dissociation constant for
N-acetylglycine at a zero [O2].
rate )
Vmax[AG][O2]
KI,AG × KM,O2 + KM,O2 × [AG] + KM,AG × [O2] + [AG][O2]
To obtain an initial protein structure consistent with the force
field, energy minimizations were performed using the conjugate
gradient algorithm in NAMD for a system solvated using the
relevant VMD44 functionality. Note, the crystal structure includes
a different peptide substrate and these coordinates were used to
place the substrates-of-interest in approximately the correct position
in the active site using a minimum least-squares overlap of peptide
coordinates. Explicit solvent was included in a cubic box with
24 490 water molecules using the TIP3P model;45 the equilibrium
density was obtained from NPT simulations at 1.0 atm and 310 K
(with Langevin therm/baro stating as implemented in NAMD).
Long-range electrostatic interactions were included via the Particle
(2)
Vmax[AG][O2]
rate )
(3)
KI,AG × KM,O2 + KM,O2 × [AG] + [AG][O2]
Predicted Active-Site Docking Conformations of N-Acyl-
glycines. The initial coordinates of the reduced PHM precatalytic
complex at 1.85 Å resolution were obtained from the Protein Data
Bank (http://www.rcsb.org/pdb/, 1SDW).13c Poses were predicted
using quantum polarized ligand docking (QPLD) to generate high
accuracy substrate binding modes utilizing molecular mechanics
(MM)36 and ab initio programs of the Schro¨dinger First Discovery
suites, Glide37 and Q-site.38
Molecular Dynamics (MD) Simulations. (i) Parameterization
and Protein Equilibration. A protein starting configuration for MD
simulations was taken from the above referenced crystal structure
(39) (a) Phillips, J. C.; Braun, R.; Wang, W.; Gumbart, J.; Tajkhorshid,
E.; Villa, E.; Chipot, C.; Skeel, R. D.; Kale, L.; Schulten, K. J. Comput.
Chem. 2005, 26, 1781–1802. (b) Sanbonmatsu, K. Y.; Tung, C. S. J.
Struct. Biol. 2007, 157, 470–480. (c) MacKerell, A. D.; et al. J. Phys.
Chem. 1998, 102, 3586–3616.
(35) The observed kinetic isotope effects reported here encompass both
the primary and R-secondary deuterium effect. Because the R-second-
ary deuterium kinetic isotope effect is relatively small (∼1.2) in
relationship to the intrinsic primary effect (10-11), the contribution
of the R-secondary effect to our measurements are within the error
(e12%).
(36) Cho, A. E.; Guallar, V.; Berne, B. J.; Friesner, R. J. Comput. Chem.
2005, 26, 915–931.
(37) (a) Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic,
J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry,
J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S. J. Med. Chem. 2004,
47, 1739–1749. (b) GLIDE; Schrodinger, LLC: Portland, OR, 2000.
(38) QSITE; Schrodinger, LLC: Portland, OR, 2000.
(40) Sousa, S. F.; Fernandes, P. A.; Ramos, M. J. J. Phys. Chem. A 2007,
111, 10439–10452.
(41) (a) Lee, C.; Yang, W. T.; Parr, R. G. Phys. ReV. B Condens. Matter
1988, 37, 785–789. (b) Lee, C.; Parr, R. G. Phys. ReV. A 1990, 42,
193–200.
(42) Stevens, W. J.; Basch, H.; Krauss, M. J. Chem. Phys. 1984, 81, 6026–
6033.
(43) Rappe, A. K.; Casewit, C. J.; Colwell, K. S.; Goddard III, W. A.;
Skiff, W. M. J. Am. Chem. Soc. 1992, 114, 10024–10035.
(44) Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graphics 1996, 14,
33–38.
(45) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.;
Klein, M. L. J. Chem. Phys. 1983, 79, 926–935.
9
J. AM. CHEM. SOC. VOL. 132, NO. 46, 2010 16397

A R T I C L E S
McIntyre et al.
Table 1. Steady-State Kinetic Constants and Deuterium Kinetic
Isotope Effects for the PHM-Mediated Oxidation on
N-Acylglycinesa b
,
N-acylglycine
substrate (AG)
(VMAX,AG
(s-1
)
)
(KM,AG)
app
(mM)
(VMAX/KAG)
app
app
R
(mM-1 s-1
)
D(VMAX/KAG
)
app
N-acetylglycine
1
2
3
5
7
9
9.2 ( 0.3 18 ( 1.6 0.51 ( 0.05 3.2 ( 0.31
N-propionylglycine
N-butyrylglycine
N-hexanoylglycine
N-octanoylglycine
N-decanoylglycine
9.8 ( 0.3 2.4 ( 0.3
11 ( 0.4 2.3 ( 0.3
2.6 ( 0.3 2.9 ( 0.39
4.6 ( 0.6 2.3 ( 0.33
11 ( 0.2 0.58 ( 0.05 18 ( 1.5 2.1 ( 0.22
13 ( 0.3 0.20 ( 0.02 66 ( 6.1 1.6 ( 0.21
13 ( 0.2 0.11 ( 0.01 130 ( 8.0 1.2 ( 0.12
a All experiments were carried out at ambient O2 meaning that the
initial O2 concentration was 217 µM. b The steady-state parameters are
reported as the experimental value ( the standard error.
Mesh Ewald method.46 Equilibration of the solvated PHM system
was determined by the convergence of root-mean-square deviation
values of the system volume to less than 1%.
Figure 2. The decrease in the D(VMAX/KAG
)app at ambient O2 (217 µM) as
(ii) Alchemical Free Energy Perturbation (AFEP) Calcula-
tions. AFEP is a dual topology hybrid molecule approach used to
calculate the relative free energy differences and was employed as
implemented in NAMD to determine the Helmholtz free energy
difference, ∆A, between two N-acylglycine substrates bound to
PHM.
the length of the N-acyl chain increases for PAM catalysis. The dashed
line is drawn solely to emphasize the linearity of these data and is not model-
based.
The relative free energy was calculated using eq 4 (below), where
∆Aafb is the Hemholtz free energy determined for each “window”
in the AFEP calculation. In eq 4, kB is Boltzmann’s constant, T is
the absolute temperature, Hb(r,p) and Ha(r,p) are the Hamiltonians
characteristic of states a and b, and <...>a denotes an ensemble
average over configurations representative of the initial state, a.
∆A ) -kT ln〈exp[-ꢀ(Hb - Ha)]〉
(4)
In this system, convergence was found to be optimal with the
windows divided into (dimensionless) intervals of 0.1 with the end
points being sampled every 0.05 to promote convergence. For each
window, 10 000 molecular dynamics equilibration steps and 50 000
ensemble averaging steps (both of 1.0 fs duration) were performed.
The alchemically permutated N-acylglycine substrates were N-
decanoyl-, N-octanoyl-, N-hexanoyl-, N-butyryl-, N-propionyl-, and
N-acetyl-. Specifically, N-decanoylglycine was chosen as the initial
state for each trial varying the final permutated ligand state to a
shorter N-acylglycine derivative. The variance over each ensemble
window was calculated in order to estimate the error of the derived
values.
Figure 3. Values for the (VMAX/KO2)app measured at a saturating concentra-
tion of the indicated N-acylglycine and 217 µM O2.
KAG,app as a function of R (∼165-fold effect) (Table 1). These
data agree nicely with those reported by Wilcox et al.29 The
primary deuterium kinetic isotope effect for the CR-H bond
cleavage for the set of N-acylglycines employed here decreased
Equilibrium dynamic NVT (310 K) simulations were performed
for all experimentally tested N-acylglycine substrates, as well as
an N-benzoylglycine control to assess the dynamic structure of the
protein substrate system.
linearly as the N-acyl chain length increased, with the D(VMAX
/
KAG app
)
decreasing from 3.21 ( 0.31 at R ) 1 to 1.24 ( 0.12
at R ) 9, respectively (Figure 2 and Table 1).
Results
The values for (VMAX/KO2)app increased linearly as the length
of the acyl chain increased from N-acetylglycine (R ) 1) to
N-decanoylglycine (R ) 9) (Figure 3). As shown in Table 2,
the (VMAX/KO2)app value increases from 37 ( 1.9 mM-1 s-1 for
N-acetylglycine to 204 ( 23 mM-1 s-1 for N-decanoylglycine,
respectively. Interestingly, the VMAX is relatively insensitive to
the acyl chain length for N-acylglycine substrates, with an
average value of 20 ( 0.7 s-1 for all the N-acylglycine substrates
included here (R ) 1-9).
Steady-State Kinetic Data and Substrate Hydrophobicity. The
(VMAX/KAG app for the PAM-catalyzed oxidation of the N-
)
acylglycines at ambient O2 exhibited a parabolic relationship
with the increase in chain length (R ) number of carbon atoms
in the linear acyl chain) (Supporting Information, Figure S1).
The (VMAX/KAG app increased over 250-fold as the N-acyl chain
)
lengthened from R ) 1 (N-acetylglycine) to 9 (N-decanoyl-
glycine), with the value from 510 ( 50 M-1 s-1 at R ) 1 to
(1.3 ( 0.08) × 105 M-1 s-1 at R ) 9, respectively (Table 1).
The increase in the VMAX,app with chain length is only ∼1.4-
Minimal Kinetic Mechanism. The N-acylglycine exhibiting
the highest D(VMAX/KAG app
at ambient O2 was N-acetylglycine,
)
fold; thus, the increase in (VMAX/KAG app results in a decrease in
)
3.2 ( 0.31 (Table 1). The minimal kinetic mechanism for
N-acetylglycine was determined by measuring the dependence
of the D(VMAX/KAG app
as a function of O2 concentration and
)
(46) (a) Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.;
Pedersen, L. G. J. Chem. Phys. 1995, 103, 8577–8593. (b) Cheatham,
T. E.; Miller, J. L.; Fox, T.; Darden, T. A.; Kollman, P. A. J. Am.
Chem. Soc. 1995, 117, 4193–4194. (c) deSouza, O. N.; Ornstein, R. L.
Biophys. J. 1997, 72, 2395–2397.
D(VMAX/KO2)app as a function of N-acetylglycine concentration.
Initial rate data were fit to the bisubstrate kinetic equations
representing either the sequential or equilibrium-ordered mech-
9
16398 J. AM. CHEM. SOC. VOL. 132, NO. 46, 2010

A R T I C L E S
a b
,
Table 2. Acyl-Chain Length Dependence of the (VMAX/KO2
)
Scheme 2. Representative Minimal Kinetic Mechanism for an
app
Equilibrium-Ordered, Sequential Mechanism for the Binding of the
N-acylglycine
substrate (AG)
(VMAX,O2
(s-1
)
)
(KM,O2
(µM)
)
(VMAX/KO2)
app
(mM-1 s-1
)
app
app
N-Acylglycine (AG) and O2 Binding to PHM (E)
R
N-acetylglycinec
N-acetylglycined
N-propionylglycine
N-butyrylglycine
N-hexanoylglycine
N-octanoylglycine
N-decanoylglycine
1
1
2
3
5
7
9
28 ( 3.3
21 ( 0.9
19 ( 0.4
17 ( 0.7
20 ( 0.6
17 ( 0.3
21 ( 0.6
550 ( 130
230 ( 30
210 ( 11
170 ( 20
150 ( 14
110 ( 7.0
100 ( 11
51 ( 7.0
92 ( 12
90 ( 5.0
99 ( 12
140 ( 13
160 ( 10
200 ( 20
cosity for both substrates over the relative microviscosity range
measured (2.07 f 5.33 ηrel). Alterations in the active site
microenvironment were observed to be chain-length independent
a For these experiments, O2 was the variable substrate with the
indicated N-acylglycine fixed at a saturating concentration, 10-15 ×
value. b The steady-state parameters are reported as the
app
as the (VMAX/KAG app term for each substrate was equal and
)
(KM,AG
)
experimental value ( the standard error. c This value for the VMAX/KO2
using N-acetylglycine as the oxidizable was calculated from a fit to the
constant over the relative viscosity range studied for both
substrates relative to the nonviscogen controls.
equilibrium-preferred equation (eq 3). d This value for the (VMAX/KO2
)
app
As the minimal kinetic mechanism of PHM for substrate
addition is sequential with the oxidizable substrate binding first
(Scheme 2), viscosity studies are a valid probe for the forward
commitment factor, cf ) (k5/k4)[1 + (k3[O2]/k2)]. The viscosity
independence of VMAX/KM for both N-acetylglycine or N-
decanoylglycine (Figure S5, Supporting Information) suggests
that binding of both substrates is in true equilibrium and the
minimal kinetic mechanism is unaltered as a function of
substrate chain length. If k2 significantly decreased as the
N-acylglycine chain length increased, the rate constant for the
chemical step (k5) would approach the value of k2 resulting in
a change in the kinetic mechanism from equilibrium-ordered
to sequential. Under these conditions, the isotope effect on the
VMAX/KM would decrease as the forward commitment increased
and would vary as a function of [O2]. The viscosity dependence
of N-acylglycine binding provides a decrease in VMAX for both
N-acetylglycine and N-decanoylglycine vs nonviscogenic con-
trols. The observed decrease in VMAX for both N-acetylglycine
and N-decanoylglycine vs nonviscogenic controls suggests that
product release (k7) is rate-limiting for PHM, consistent with
the lack of a VMAX isotope effect (Table 3).
using N-acetylglycine as the oxidizable was measured by varying the
initial [O2] at saturating [N-acetylglycine].
Table 3. Deuterium Kinetic Isotope Effects for N-Acetylglycine
Oxidationa
oxidizable substrate
parameter
N-acetylglycine
[2H2]-N-acetylglycine
DKIE
VMAX (s-1
)
28 ( 3.3
51 ( 7.0
17 ( 3.0
22 ( 1.3
19 ( 0.7
14 ( 0.7
1.3 ( 0.2
2.7 ( 0.4
1.2 ( 0.2
VMAX/KO2 (mM-1 s-1
KI,AG (mM)
)
a The deuterium isotope effects (DKIE) for each relevant kinetic
parameter ((standard error) are included.
anisms (eqs 2 and 3), respectively. The data best fit an
equilibrium-ordered kinetic mechanism (eq 3) with σ values of
0.27 (H) and 0.13 (D) and variance values 0.074 (H) and 0.016
(D), respectively. By comparison, data fit to the sequential
kinetic mechanism had comparable σ and variance values though
many calculated parameters were negative and had a high error
suggesting a lack of significance for many of the terms.
The magnitude of the D(VMAX/KAG) term was constant, 1.9 (
0.2 (Supporting Information, Table S1), though lower than the
Furthermore, the viscosity studies directly probe the relative
“stickiness” of the N-acylglycine to PHM. The lack of a
microviscosity effect on the VMAX/KM values for both N-
acetylglycine and N-decanoylglycine means that the ratio of the
VMAX/KM values for the two substrates are microviscosity
D
observed (VMAX/KO2) value of 2.7 ( 0.4 (Table 3). Replot of
D
the (VMAX,AG app,
)
vs [O2] to determine the (VMAX) (Supporting
Information, Figure S2), yielded VMAX values of 21 ( 1.6 s-1
for N-acetylglycine and 24 ( 2.0 s-1 for [R-2H2]-N-acetylglycine
giving a D(VMAX) of 0.88 ( 0.10. These values are in reasonable
agreement with those obtained by a fit of the kinetic data to the
rate equation for an equilibrium-ordered mechanism (eq 3):
VMAX values of 28 ( 3 s-1 (H) and 22 ( 1.4 s-1 (D)
N-acetylglycine with a D(VMAX) of 1.3 ( 0.2 (Tables 2 and 3).47
It should also be noted that the D(VMAX/KO2) calculated by
replot analysis yielded a value of 2.5 ( 0.6 (Supporting
Information, Figure S2). These data show apparent terms for
independent, despite the differences in the D(VMAX/KAG app
)
values: 3.2 for N-acetylglycine and 1.2 for N-decanoylglycine.
The decrease in the D(VMAX/KAG app
)
with acyl chain length cannot
be attributed to an increased forward commitment factor because
the k5/k2 ratio does not change enough to perturb the equilibrium.
Using this rationale, it can be concluded that all the N-
acylglycines share an equilibrium-ordered mechanism, where
k2 is much greater than k5, as well as k3,O2[O2] (Scheme 2).
Therefore, the observed KIEs (Figure 2) cannot be attributed
to a change in the minimal kinetic mechanism for this library
of substrates. An equilibrium-ordered kinetic mechanism for
N-decanoylglycine is supported by the [O2]-independence of the
the D(VMAX/KAG app
kinetic isotope effect to be constant as a
)
function of oxygen concentration. The dissociation constant for
the N-acetylglycine substrate, KI,AG, was independent of R-car-
D
bon deuterium substitution with KI,AG values of 1.2 ( 0.2.
Viscosity Effects. Values measured for both VMAX and VMAX
D(VMAX/KAG app
shown).
)
when measured from 30-200 µM O2 (data not
/
KM as a function of Ficoll-400 concentration (the macrovisco-
gen) show no deviation from controls for the oxidation of either
N-acetylglycine or N-decanoylglycine, respectively. Increased
microviscosity resulted in a decreased rate of product release
(VMAX) for N-acetylglycine and N-decanoylglycine. However,
Computational Chemistry. (i) Predicted N-Acylglycine Dock-
ing Conformations. Our docking results indicate that the N-
acylglycine substrates bind into a hydrophobic pocket within
the known PHM active site in the ternary AG ·O2 ·enzyme
complex. The N-acylglycine substrates are observed to form
an ionic bond between the carboxylate of the substrate glycyl
residue and the guanidine group of Arg240. This ionic interaction
between substrate carboxylate and Arg240 has also been observed
in the crystallized PHM structure (1SDW) containing bound
(VMAX/KAG app showed no significant dependence on microvis-
)
(47) The VMAX,app at one fixed [O2] and the D(VMAX/KAG) value had to be
determined from a replot of VMAX,app vs [O2] because the rate equation
for an equilibrium-ordered kinetic mechanism is unsymmetrical.
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A R T I C L E S
McIntyre et al.
acetylglycine molecule performed an inversion within the PHM
active site during the simulation, consistent with docking
orientation poses predicted (Supporting Information, Figure S3).
This phenomenon was partially observed during the AFEP
studies, as the Arg240 salt bridge was not observed for the
“decanoylglycine-to-acetylglycine” AFEP-transformation as the
final state was achieved. For the other substrates, the position
of the acyl chain within the active site was modulated by the
hydrophobic pocket. Note that equilibrium dynamic (MD)
simulation of binding of N-benzoylglycine to PHM displayed
no deviation in the salt bridge between Arg240 and the carbox-
ylate of the glycyl moiety. This was similar to the MD
simulation for the binding of N-decanoylglycine, although we
did observe greater movement of the benzene ring. N-Ben-
zoylglycine (hippurate) has long been known to be a PHM
substrate, but with a relatively low (VMAX/Khippurate app value
)
relative to peptide and longer chain N-acylglycine substrates.29,48
The CR-H T O2 distance minima achieved during the simula-
tions of all the N-acylglycine derivatives approaches the sum
of the van der Waals radii for oxygen and hydrogen. The
classical force field is not capable of pushing the distances any
closer than this without external forces being applied. Note that
our intent is to determine the free energy of binding and not to
simulate the actual reaction. Other methods are better suited
for such a task, for example, QM/MM.
Figure 4. A plot of the ∆GALCHEMICAL vs N-acylglycine chain length. For
the above molecular dynamics (MD) simulations, relative free energies were
calculated with N-decanoylglycine treated as the initial state (λ ) 0) with
respect to shorter chain N-acylglycine substrates (λ ) 1). Because of the
“decanoylglycine-to-acetylglycine” transformation appearing below the
expected trend, “butyrylglycine-to-propionylglycine” and “butyrylglycine-
to-acetylglycine” transformations were also performed and plotted (open
circles).
N-acetyl-diiodo-tyrosyl-D-threonine.13 Of the N-acylglycines
investigated computationally, all of the substrates except N-
acetylglycine interacted with an active site, hydrophobic pocket
comprised of the residues Leu206, Met208, Ile306, and Met314
(Supporting Information, Figure S3). The lack of interaction
between N-acetylglycine and the hydrophobic pocket can be
attributed to the size of the substrate as the Euclidean distance
between Arg240, the site of ionic interaction, and the hydrophobic
pocket is simply too great. The strength of this interaction
increases as a function of increasing substrate N-acyl chain
length. These ionic and hydrophobic interactions were observed
to be the predominant determinants for substrate binding when
forming the ternary reduced PHM ·acylglycine·O2 complex in
our computational investigation.
Discussion
Minimal Kinetic Mechanism for the PHM Mediated
Oxidation of the N-Acylglycines. Previous work has established
that the addition of the oxidizible substrate occurs after the
reduction of enzyme-bound Cu(II) atoms.18e,19 Using ascorbate
as the exogenous reductant, Merkler et al.49 demonstrated that
the two electrons required for the reduction of the PHM-bound
Cu(II) atoms are delivered from two one-electron reduction
steps, resulting in the oxidation of two ascorbate molecules to
two semidehydroascorbate molecules per enzyme turnover.
Reduction of PHM-bound Cu(II) is not included in the minimal
mechanism as the oxidized reductant dissociates following
reduction.18b The initial rate patterns for both N-acetylglycine
and [R-2H2]-N-acetylglycine are most consistent with an equi-
librium-ordered mechanism (Scheme 2): the convergence of
1/initial rate vs 1/[N-acetylglycine] at increasing [O2] in the
second quadrant while the 1/initial rate vs 1/[O2] at increasing
[N-acetylglycine] intersecting at the abscissa (Supporting In-
formation, Figure S4).50 An equilibrium-ordered mechanism can
(ii) Alchemical Free Energy Perturbation (AFEP) and Equi-
librium Dynamics. The AFEP simulations resulted in a thermo-
dynamic plot of ∆G vs R which illustrates the inverse
relationship between relative dissociation energy (∆GALCHEMICAL
)
and chain length, increasing and decreasing respectively for
N-acylglycine substrates which were observed to interact with
the hydrophobic pocket. At R ) 1 (N-acetylglycine), the
∆GALCHEMICAL value is ∼24 kcal/mol lower than the R ) 2 (N-
propionylglycine) complex and ∼12 kcal/mol lower than R )
9 (N-decanoylglycine) complex, the most stable complex
identified from the set of N-acylglycines employed in our work
(Figure 4). The decrease in ∆GALCHEMICAL vs acyl chain length
indicates that increasing the hydrophobicity of the N-acylglycine
substrates increases the stability of the AG ·O2 ·enzyme complex.
In other words, dissociation of the N-acylglycine from
AG·O2 ·enzyme to form the binary O2 ·enzyme + AGfree
becomes increasingly favorable as the length of the acyl chain
decreases.
be differentiated from equilibrium random as the replot of (KAG
/
VMAX app
)
against 1/[O2] passes through the origin.50,51 Therefore,
the order of substrate addition to PHM was sequential with the
addition of the N-acylglycine followed by O2 to form the central
complex, PHM-2Cu(I)·AG·O2. An equilibrium-ordered mech-
anism is consistent with previous studies of PAM and
PHM.18e,19,52 As the rate expression for an equilibrium-ordered
kinetic mechanism is unsymmetrical (eq 3),50,51 a signature for
this mechanism becomes equivalent magnitudes of the D(VMAX
/
The equilibrium dynamics calculations indicate a drastically
altered N-acetylglycine pose compared with all other N-
acylglycine substrates. The goal of our NPT simulation was to
sample each N-acylglycine substrate conformation in the absence
of an alchemical permutation (AFEP). Over the 1 ns simulation,
N-acetylglycine (R ) 1) sampled many more binding conforma-
tions than the substrates with longer acyl chains. The N-
(48) Katopodis, A. G.; May, S. W. Biochemistry 1990, 29, 4541–4548.
(49) Merkler, D. J.; Kulathila, R.; Consalvo, A. P.; Young, S. D.; Ash,
D. E. Biochemistry 1992, 31, 7282–7288.
(50) Cook, P. F.; Cleland, W. W. Biochemistry 1981, 20, 1790–1796.
(51) Cook, P. F.; Cleland, W. W. Enzyme Kinetics and Mechanism; Garland
Science Publishing: New York, NY, 2007.
(52) Francisco, W. A.; Wille, G.; Smith, A. J.; Merkler, D. J.; Klinman,
J. P. J. Am. Chem. Soc. 2004, 126, 13168–13169.
9
16400 J. AM. CHEM. SOC. VOL. 132, NO. 46, 2010

A R T I C L E S
KM) values for both the N-acylglycine substrates and O2.50,53
Experimentally, this is exactly what we found as (VMAX/KM)
The thermodynamics obtained from the AFEP calculations
only provide insight about the relative stability of the optimal
conformation for the individual reduced PHM ·acylglycine·O2
complexes once these have been achieved. Conversely, the
steady-state kinetics and the KIEs describe the probability of
reaching the most stable (or optimal) conformation. Increasing
the chain length promotes the interaction between the acyl chain
and the hydrophobic pocket adjacent to the CuM domain
(Supporting Information, Figure S3) and modulate the fidelity
of PHM catalysis. The viscosity effects suggested that the
association rate constant for N-acylglycine binding was similar
for all the substrates. This is coupled to the fact that the
magnitude of the dissociation rate constant becomes smaller as
the acyl chain length gets longer, suggesting that that the
Kdissociation () koff/kon) would also decrease as the acyl chain
lengthens. Therefore, the ∆G°dissociation would become more
positive and less favorable as the acyl chain of the substrates
becomes longer. The AFEP analysis correlates well with the
relative free energy for CR-H cleavage, providing an internal
probe for both the chemistry and binding steps. The dissociation
rate constant for the N-acylglycines decreases as the acyl chain
lengthens and is perturbed directly into the rate constant for
the chemical step (CR-H cleavage) such that the k5/k2 ratio does
not change enough to alter the kinetic mechanism. The lack of
a microviscosity effect on the VMAX/KM values for both
N-acetylglycine and N-decanoylglycine and [O2]-independence
of D(VMAX/KM) for N-decanoylglycine is also consistent an
equilibrium-ordered minimal kinetic mechanism for all N-
acylglycine substrates included here. Overall, the combination
of kinetic and thermodynamic data presented in this study
suggests that transient dynamic motions of PHM favor the more
hydrophobic N-acylglycines to more efficiently sample con-
former distances for optimal wave overlap between CR-H and
Cu/O2 as substrate orientation becomes increasingly constrained
proportional to chain length.
D
for N-acetylglycine and O2 were equivalent within experimental
error, 1.9 ( 0.2 for N-acetylglycine and 2.0 ( 0.1 for O2 (Tables
S1 and 3). The equilibrium-ordered mechanism is rationalized
with k2,AG being much faster than k3,O2[O2] at any [O2] (see
Scheme 2).18e,51 As shown in eq 5, the (VMAX/KM) value for
D
each substrate in the equilibrium-ordered mechanism is reduced
to the following:
k5
Dk5 +
Vmax
KM
k4
Vmax
KM
D
D
)
)
(5)
(
)
(
)
k5
AG
O2
1 +
k4
The binding of N-acetylglycine to reduced PHM is in
equilibrium, such that the off-rate from the E ·acetylglycine
complex, k2, is much greater than the rate of catalysis, k5
(Scheme 2).51 The microviscosity data show no acyl chain length
dependence (Supporting Information, Figure S5). Therefore, the
∼250-fold increase in (VMAX/KAG app
)
as the acyl chain length
increases from N-acetylglycine to N-decanoylglycine (Table 1)
catalytic cannot be attributed to either a fully or partially
diffusion-limited process.54 This eliminates the possibility of
the equilibrium-ordered mechanism found for N-acetylglycine
addition to PAM changing to a sequential mechanism for the
other N-acylglycine substrates as chain length increases. For
D
an equilibrium-ordered mechanism, the magnitude of (VMAX
/
KAG) would differ from the D(VMAX/KO2) as the former term
would become dependent on [O2], while the latter would remain
constant as a function of [N-acylglycine].51
Acyl Chain Length Dependence of the Binding Mode. The
plot of ∆G vs chain length (R) shows that dissociation energy
(∆GALCHEMICAL) increases as R decreases from 9 to 2. At R ) 1,
the ∆G values were ∼24 kcal/mol lower than that for R ) 2 and
∼12 kcal/mol lower than that for R ) 9, the most stable complex
included in our study (Figure 4). These results are derived from
the AFEP calculations by direct comparison with N-decanoylgly-
cine (R ) 9). Each N-acylglycine is bound in the PHM active site
in the same orientation, ionic bonding between the carboxylate and
Arg240, and an interaction between the acyl chain and the
Comparison of the R-carbon donor position between both
N-benzoylglycine and N-decanoylglycine shows very little
difference in the orientation of these atoms over the course of
the MD simulation for the glycyl moiety (Supporting Informa-
tion, movie files). The benzoyl group was observed to display
a much greater degree of conformational sampling compared
with the decanoyl group. The VMAX/KM and KM values for
N-decanoylglycine are 25-fold higher and 13-fold lower than
those for N-benzoylglycine.29 The role of Arg240 in protein gating
has been addressed with an R240Q PHMcc mutant, with a 2-fold
hydrophobic pocket defined by Leu206, Met208, Ile306, and Met314
.
The exception to this was N-acetylglycine (Figure 4). The
anomalous behavior of N-acetylglycine behavior could be attributed
to the configurational ensembles not having a large degree of the
desired overlap between states, thereby not providing the requisite
accuracy during the AFEP permutations.39a For the substrate range
N-propionylglycine through N-decanoylglycine (R ) 2 f 9), the
increased acyl chain length of the substrate was proportional to an
increase in the dissociation energy of substrate from the central
complex (Figure 4). The ∆GALCHEMICAL vs chain length plot, based
on the AFEP calculations, demonstrated that increasing the
hydrophobicity of the N-acylglycine substrate results in a propor-
tional increase in the ∆Gdissociation for the dissociation of the substrate
from the reduced PHM·acylglycine·O2 complex. In other words,
as the acyl chain lengthens, the N-acylglycine substrate is less likely
to dissociate from the reduced PHM·acylglycine·O2 complex.
increase in KM and a 200-fold decrease in VMAX.
13a Results from
this mutation suggest that Arg240 functions to pose a geometrical
constraint upon both the oxidizable substrate and protein
assisting the Franck-Condon gating dynamics to optimize
hydrogen donor-acceptor distances. An advantage for hydro-
phobic substrates appears to be an extra stabilization provided
by interaction with the hydrophobic pocket within the PHM
active site which is used to reach the transfer configuration more
effectively. Therefore, the increased contact points between
enzyme and the N-acylglycine substrates as the acyl chain
increases in hydrophobicity most likely assists frequency
modulation between the hydrogen donor and Cu(II)-superoxo
acceptor to more easily to achieve degenerate states.
Conclusion. The kinetic, viscosity, and KIE data included herein
show that N-acylglycine binding to PHM is in equilibrium before
catalysis can occur and that the dissociation of the N-acylglycine
from the reduced PHM ·S becomes less favorable as the length of
the acyl chain increases. With the exception of N-acetylglycine,
(53) (a) Cook, P. F. Isotopes EnViron. Health Stud. 1998, 34, 3–17. (b)
Evans, J. P.; Blackburn, N. J.; Klinman, J. P. Biochemistry 2006, 45,
15419–15429.
(54) (a) Stone, S. R.; Morrison, J. F. Biochemistry 1988, 27, 5493–5499.
(b) Brouwer, A. C.; Kirsch, J. F. Biochemistry 1982, 21, 1302–1307.
9
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A R T I C L E S
McIntyre et al.
the MD simulations indicate that each N-acylglycine binds to
reduced PHM in approximately the same orientation: an ionic
interaction between the substrate carboxylate and Arg240 and an
interaction between the acyl chain and an active site hydrophobic
pocket (Supporting Information, Figure S3).
domain of bifunctional PAM. Future studies will address this most
interesting possibility.
Given the mechanistic similarities between PHM and DꢀM,
preorganization is likely to be important for DꢀM catalysis as well.
In fact, an active site glutamine residue in DꢀM, Gln369, may have
a role similar to the active site hydrophobic pocket in PHM as the
The intent of the AFEP poses was to elucidate the contribution
of acyl chain length to the relative dissociation energies for each
ligand, benchmarked to the most hydrophobic substrate N-
decanoylglycine. The equilibrium molecular dynamics simula-
tions showed decreased active site sampling associated with
increased chain length. The minima achieved during the
simulations of all N-acylglycine derivatives approaches the van
der Waals radii for oxygen and the CR-hydrogen. The classical
force field is incapable of creating distances any closer than
this without the application of external force(s). The utilization
of a hydrophobic pocket in the active site and carboxylate-Arg240
salt bridge for substrate positioning in the PHM ternary complex
was the mechanism by which CR-H activation became decreas-
ingly rate determining. The decrease in the KIE with hydro-
phobicity (Figure 2) suggests that environmentally coupled
tunneling phenomena were more efficient as the probability of
optimal conformer sampling increased due to the N-acylglycine
hydrophobic pocket and Arg240 interaction. The role of the active
site hydrophobic pocket and Arg240 appear to preorganize the
bound substrate allowing protein promoting (gating) vibrations
to assist the through-barrier tunneling process. Overall, preor-
ganization of the N-acylglycine substrate facilitated by the
hydrophobic pocket and Arg240 provide a greater probability of
a transient reduced PHM ·S·O2 complex to be productive in
the reaction coordinate. The chain length dependence for
preorganization suggests that optimal overlap for acceptor and
donor moieties can be regulated by both enzyme dynamics and
substrate structure. In this sense, substrate hydrophobicity lends
itself to a synergistic event increasing the probability of a
hydrogen tunneling event.
3,4-dihydroxbenzene moiety of dopamine bonds weakly with
55
Gln369
.
In addition, the fumarate-dependent activation of DꢀM
could be analogous to the role of substrate carboxylate-Arg240 salt
bridge in PHM. It has been proposed that fumarate activation was
the result of reduced phenylethylamine motion (preorganization)
due to a weak interaction between anionic fumarate and the primary
amine.56 In sum, the observations of this study describe the active
site of PHM as extremely well designed to accommodate the spatial
requirements for donor and acceptor wave overlap. The decrease
in the KIE with hydrophobicity (Figure 4) suggest that environ-
mentally coupled tunneling phenomena become more efficient as
the probability of optimal conformer sampling increased due to
interactions between the N-acylglycine and both the hydrophobic
pocket and Arg240
.
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. & William L. Griffin
Foundation, the Alpha Research Foundation, Inc., the Eppley Founda-
tion for Research, the Gustavus and Louise Pfieffer Research Founda-
tion, 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., financial support from Louisiana Cancer Research Consor-
tium (LCRC), Center for Undergraduate Research (CUR) and Research
Centers in Minority Institutions (RCMI) to N.R.M, and a predoctoral
fellowship to E.W.L. from the American Heart Association (0415259B).
This project was supported by an allocation from the TeraGrid
Advanced Support Program (TG-MCB070112N) and services of
Research Computing at the University of South Florida. The authors
dedicate this work to the memory of Dr. Terence C. Owen.
Francisco et al.23 suggest that H · tunneling in PHM catalysis
was dominated by gating motions. Their results are not inconsistent
with the data presented here as Francisco et al.23 did not argue
that preorganization had no role in PHM stunneling. However, there
are some other intriguing differences between our work and that
of Francisco et al.23 The substrate used by Francisco et al.52 was
hippurate and our MD simulations show highly randomized
movement of the benzene ring and, thus, preorganization may be
less important for this PHM substrate. Another difference is our
use of bifunctional PAM to study PHM while Francisco et al.23
studied monofunctional PHM. It is possible that PAL may modulate
the dynamics of the PHM domain such that preorganization plays
a more important role in H · tunneling as catalyzed by the PHM
Supporting Information Available: Data referred to in the
text, NMR spectra for the N-acylglycines synthesized for this
work, and complete lists of authors for refs 29, 30, and 39c.
This information is available free of charge via the Internet at
http://pubs.acs.org.
JA1019194
(55) (a) Kamachi, T.; Kihara, N.; Shiota, Y.; Yoshizawa, K. Inorg. Chem.
2005, 44, 4226–4236. (b) Yoshizawa, K.; Kihara, N.; Kamachi, T.;
Shiota, Y. Inorg. Chem. 2006, 45, 3034–3041.
(56) Ahn, N.; Klinman, J. P. Biochemistry 1983, 22, 3096–3106.
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16402 J. AM. CHEM. SOC. VOL. 132, NO. 46, 2010