Pre-steady-state kinetic and structural analysis of interaction of methionine γ-lyase from Citrobacter freundii with inhibitors

Article abstract of DOI:10.1074/jbc.M114.586511

Methionine γ-lyase (MGL) catalyzes the γ-elimination of L-methionine and its derivatives as well as the β-elimination of L-cysteine and its analogs. These reactions yield α-keto acids and thiols. The mechanism of chemical conversion of amino acids includes numerous reaction intermediates.The detailed analysis of MGL interaction with glycine, L-alanine, L-norvaline, and L-cycloserine was performed by pre-steady-state stopped-flow kinetics. The structure of side chains of the amino acids is important both for their binding with enzyme and for the stability of the external aldimine and ketimine intermediates. X-ray structure of the MGL·L-cycloserine complex has been solved at 1.6 A? resolution. The structure models the ketimine intermediate of physiological reaction. The results elucidate the mechanisms of the intermediate interconversion at the stages of external aldimine and ketimine formation.

Full text of DOI:10.1074/jbc.M114.586511

Enzymology:
Pre-Steady-State Kinetic and Structural
Analysis of Interaction of Methionine γ
-Lyase from Citrobacter freundii with
Inhibitors
Nikita A. Kuznetsov, Nicolai G. Faleev,
Alexandra A. Kuznetsova, Elena A.
Morozova, Svetlana V. Revtovich, Natalya V.
Anufrieva, Alexei D. Nikulin, Olga S.
Fedorova and Tatyana V. Demidkina
J. Biol. Chem. published online November 14, 2014
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Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
Pre-Steady-State Kinetic and Structural Analysis of Interaction of Methionine γ-Lyase from
Citrobacter freundii with Inhibitors*
Nikita A. Kuznetsov1,2, Nicolai G. Faleev3, Alexandra A. Kuznetsova1,2, Elena A. Morozova4,
Svetlana V. Revtovich4, Natalya V. Anufrieva4, Alexei D. Nikulin5, Olga S. Fedorova1,2,§, Tatyana V.
Demidkina4,§
1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of
Sciences, Novosibirsk 630090, Russia
2Department of Natural Sciences, Novosibirsk State University, Novosibirsk 630090, Russia
3Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Moscow 119991,
Russia
4Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow 119991, Russia
5Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142290,
Russia
*Running title: Kinetic and Structural Studies of Inhibitors of Methionine γ-Lyase
§To whom correspondence should be addressed: O.S.F. Tel: +7(383)363 5174, Email:
fedorova@niboch.nsc.ru; T.V.D. Tel.: +7(499)135 9858, Email: tvd@eimb.ru
Keywords: Pyridoxal 5'-phosphate dependent methionine γ-lyase, competitive inhibitors, pre-steady-state
kinetics, spatial structure of pyridoxal 5'-phosphate-cycloserine derivative.
Background: Speculative chemical mechanism of
methionine γ-lyase is formulated, kinetic and
structural data concerning elementary stages of
physiological reaction are mostly lacking.
Results: Pre-steady-state kinetic and structural
analysis of the enzyme interaction with inhibitors
was performed.
and ketimine intermediates. X-ray structure of
MGL-L-cycloserine complex has been solved at
1.6 Å resolution. The structure models ketimine
intermediate of physiological reaction. The
results elucidate the mechanisms of the
intermediates interconversion at the stages of
external aldimine and ketimine formation.
Conclusion: Results elucidate mechanism of
intermediates interconversion at initial stages of
enzymatic reaction.
Significance: The data serve for understanding
detailed mechanism of pyridoxal 5′-phosphate
dependent γ-elimination reaction.
Methionine γ-lyase (EC 4.4.1.11, MGL), a
pyridoxal 5'-phosphate (PLP) dependent enzyme,
is found in many bacteria (1) including
Enterobacteriaceae family (Citrobacter freundii
(2)) and in pathogenic bacteria (Bacteroides ssp.
(3), Aeromonas sp. (4), Clostridium sporogenes
(5), Porphyromonas gingivalis (6), Treponema
denticola (7)). MGL has also been found in
parasitic eukaryotes (the protozoa Entamoeba
histolytica (8) and Trichomonas vaginalis (9)) and
in a plant Arabidopsis thaliana (10).
The absence of the enzyme in mammals allows
MGL to be considered as a drug target for the
treatment of infectious diseases. In addition, MGL
has been utilized to develop the therapeutic
treatment of tumors, by introducing recombinant
proteins to deplete methionine, which is essential
for the growth of cancer cells (11-13).
ABSTRACT
MGL catalyzes the γ-elimination of L-
methionine and its derivatives as well as the β-
elimination of L-cysteine and its analogs. These
reactions yield α-keto acids and thiols. The
mechanism of chemical conversion of amino
acids
includes
numerous
reaction
intermediates. The detailed analysis of MGL
interaction with glycine, L-alanine, L-norvaline
and L-cycloserine was performed by pre-
steady-state
stopped-flow
kinetics.
The
The biological unit of MGL is a tetramer which
can be subdivided into two so-called catalytic
dimers. Every dimer contains two active sites
structure of side chains of the amino acids is
important both for their binding with enzyme
and for the stability of the external aldimine
1
Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
consisting of amino acid residues from the both
subunits and two molecules of PLP covalently
bound to Lys210 (14).
norvaline and L-cycloserine. The stopped-flow
kinetic analysis of the single-wavelength
absorbance permitted us to attribute them
separately to particular intermediates of the
reaction. X-ray structure, modeling the ketimine
intermediate of the γ-eliminating reaction, has
been solved at 1.6 Å resolution. These data will
serve for elucidation of mechanisms of
physiological reaction catalyzed by C. freundii
MGL and can be helpful for a design of new
inhibitors of MGL as potential drugs for a
treatment of infection diseases.
MGL catalyzes the irreversible γ-elimination of
L-methionine to give methanethiol, α-ketobutyrate
and ammonia (Eq. 1). The enzyme is also able to
catalyze β-elimination reaction of L-cysteine and
S-substituted L-cysteines (Eq. 2) and γ- and β-
replacement reactions of L-methionine and L-
cysteine and their analogs (15,16). The chemical
mechanism (Scheme 1) of γ-elimination reaction
was proposed by Brzovic (17).
EXPERIMENTAL PROCEDURES
Materials, amino acids, enzymes
chemicals were from Sigma-Aldrich (St Louis,
MO).
–
All
(1)
The recombinant MGL was obtained from
Escherichia coli BL 21 (DE3) cells containing
pET-mgl plasmid with the inserted megL gene
from C. freundii genome. Growing of the cells and
purification of the enzyme was carried out as
described previously (2). Protein concentrations
were determined by the method of Lowry et al.
(22), using bovine serum albumin as a standard.
Activity of the enzyme was assayed by measuring
the rate of α-ketobutyrate formation from L-
methionine by the method of Friedemann and
Haugen (23). One unit of enzymic activity was
determined as the amount of enzyme catalyzing
transformation of 1 μmol of L-methionine per min
at 30 °C. The specific activity of MGL was 8.5
u/mg.
(2)
The initial stages of the γ-elimination occur by
the exchange of -amino group of Lys210 in
internal aldimine (I) to α-amino group of L-
methionine through the fast formation of the
geminal diamine (II) and its following conversion
to the external aldimine (III). In the external
aldimine (III) the proton is abstracted from the α-
carbon atom of substrate and a quinonoid
intermediate (IV) is formed. Subsequent
protonation of the C4'-atom of the coenzyme and
abstraction of a Cβ-proton of the substrate leads to
the formation of ketimine (V) and enamine (VI)
intermediates. The elimination of the thiol group,
the sequential formation of β,γ-unsaturated
ketimine (VII), α-aminocrotonate (VIII) and
hydrolysis of the Schiff base in α-aminocrotonate
lead finally to release of α-keto acid and ammonia.
Intermediates of the γ-elimination reaction
catalyzed by PLP-dependent enzymes possess the
distinct absorption spectra (18).
Despite of spectral and structural information
concerning MGL (14,19-21) the kinetic
mechanisms of β- and γ-elimination reactions
catalyzed by the enzyme remain poorly
understood. Therefore, the detailed analysis of the
changes in the absorption spectra accompanying
the binding of the amino acids allows us to
elucidate the mechanisms of the intermediates
interconversion.
Pre-steady state stopped-flow studies
–
Stopped-flow measurements with absorption
detection were carried out using a model SX20
stopped-flow spectrometer (Applied Photophysics,
U.K.) with a 150 W Xenon lamp and 10 mm
pathlength optical cell. The dead time of the
instrument was 1.0 ms. All experiments were
carried out at 25 °C in 0.1 M potassium phosphate
buffer solution (pH 7.8), containing 0.5 mM DTT
and 0.1 mM EDTA. Solutions of enzyme (12.5
µM) were mixed with various concentrations of
glycine (10-500 mM), L-alanine (1.0-12.0 mM),
L-cycloserine (6.35-38.1 µM), and L-norvaline
(1.5-7.5 mM). Each kinetic curve was averaged
over at least three independent experiments. The
absorbance at 320, 420 and 500 nm was detected.
Kinetic data analysis – Estimation of the
kinetic mechanisms as well as the number of
individual reaction steps was implemented as
follows. Each kinetic trace was fitted by sum of
In the present work we have studied the kinetic
mechanisms of binding of MGL from C. freundii
with competitive inhibitors, glycine, L-alanine, L-
2

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
the exponential to define observed rate constants
kobs.
Steady state kinetics – Inhibition of L-
methionine γ-elimination reaction by L-
cycloserine was determined in the reaction
mixtures containing 100 mM potassium phosphate
buffer (pH 8.0), 0.1 mM PLP, 1 mM EDTA, 5
mM DTT, and varied amounts of the substrate and
the inhibitor. The rate of α-ketobutyrate
Dc = A1×exp(−kobs1×t) + A2×(1 − exp(−kobs2×t)) +
Db
Dc is the absorbance intensity at any reaction time
t; A1, A2 are the amplitudes; kobs1, kobs2 are the
observed rate constants; Db is the background
absorbance.
Values of the observed rate constants kobs were
plotted versus amino acid concentrations in order
to estimate the individual rate constants. As an
example the concentration dependence of kobs for
the fast and slow phases at 320 nm for L-Ala are
accumulation
was
determined
using
dinitrophenylhydrazine (23). The reaction was
initiated by addition of the enzyme (15 μg). The
mixture was incubated for 15 min at 30°C, and the
reaction was stopped by addition of trichloroacetic
acid to the final concentration of 12.5% (w/v).
Analysis of the data was processed by Dixon (29)
and Lineweaver and Burk plots (30) and inhibition
constant was determined using the EnzFitter
program.
shown in Supplementary Figure S1. The fast phase
1
fits a straight line according to the function kobs
=
Crystallization and data collection – Crystals
of MGL were obtained in the same conditions as
described in (31) using polyethylene glycol
monomethyl ether 2000 as the precipitant. The
complex of MGL with L-cycloserine was obtained
by soaking of holoenzyme crystals in
cryoprotective mother liquid solution, containing
L-cycloserine (50 mM, 7 min). The diffraction
data were obtained at the BW6B DESY beamline
with MAR345 MAResearch Image Plate. The
detailed data collecting statistics are shown in
Table 1.
Structure determination and refinement – The
structure was solved by molecular replacement
using the structure of C. freundii MGL (PDB ID
2RFV) by rigid body procedure, implemented in
CCP4 software suite (32).
k1×[L-Ala]+k-1. The slow phase fits the function
2
kobs = Kb×[L-Ala]×k2/(Kb×[L-Ala]+1)+k-2. The
i
dependences of kobs on L-Ala concentration allow
to estimate all individual rate constants
(Supplementary Figure S1), which were
subsequently used as initial values in a global
analysis.
Global non-linear least-squares fitting was
performed using DynaFit4 software (BioKin Ltd)
(24) as described in (25-28). Differential equations
were written for each species in the mechanisms
described in Schemes 3 and 4 (see Results and
Discussion), and the stopped-flow absorbance
traces were directly fit by expressing the
absorbance intensity (Dc) at any reaction time t as
the sum of the background absorbance (Db) and
the absorbances of each ith intermediate Di(t):
n
The model was improved using manual
rebuilding with a COOT (33) and maximum
likelihood refinement using REFMAC5 (34).
Flexible loops of the protein and water molecules
were removed from the initial model to exclude
model bias during the first round of refinement.
The presence of covalently bound to the PLP
cycloserine molecule is clearly visible in the
experimental 2Fo-Fc and Fo-Fc electron density
maps. The final model also contains a chloride ion,
266 water and 4 polyethylene glycol monomethyl
ether 2000 molecules. The structure has been
deposited to the Protein Data Bank with PDB ID
4OMA.
D  D  D (t)

c
b
i
i0
where Di(t) = εi[Ei(t)], εi are the extinction
coefficients at the specific wavelength for each
discernible intermediate, and [Ei(t)] are the
concentrations of this intermediate at any given
time t (i = 0 relates to the free protein; i > 0 relates
to the protein–inhibitor complexes).
In the data processing, global fits of sets of
kinetic traces obtained at different concentrations
of the reactants were done to derive the relevant
kinetic scheme and to determine the kinetic
parameters. In the fits all rate constants (including
rate constants for the forward and backward
reactions) as well as the specific molar extinction
coefficients for all intermediate complexes were
optimized.
RESULTS AND DISCUSSION
Reaction of methionine γ-lyase with glycine – It
was shown that MGL catalyzes exchange of both
Cα-protons of glycine with high stereospecificity
3

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
for (pro)-R-proton which reacts by a factor of
namely this proton. On the other hand, complex
(E•Gly)3, which also absorbs at 420 nm, but is
formed much slower, may correspond to the
external aldimine in other conformation, with an
orthogonal orientation of the ‘wrong” pro-(S)-
proton (Hw) (IIIb, Fig. 2). This conformation is
suitable for the exchange of pro-(S)-proton, while
exchange of the pro-(R)-proton is not possible.
The observed rate of pro-(S)-proton exchange is
slower than the rate of (E•Gly)3 formation, so, in
principle, this complex may be an intermediate in
the isotopic exchange reaction.
1440 faster than does pro-(S)-proton (35). In
principle the exchange may proceed through the
formation of a quinonoid intermediate, or by a
concerted mechanism implying fast and reversible
transfer of the (pro)-R-proton from Cα-atom to
C4'-atom, or a formation of a six-membered
transition state incorporating a water molecule
(36) (Scheme 2). To assess the formation and
decay of individual intermediates which are
formed in the reaction of MGL with glycine, we
carried out single-wavelength stopped-flow
experiments at 320, 420 and 500 nm.
In accordance with the proposed scheme the
external aldimine is rearranged from conformation
IIIa to conformation IIIb through the formation of
an intermediate complex (E•Gly)2, absorbing at
320 nm. Probably most possible structure of
complex (E•Gly)2 may be the ketimine, whose
formation can be affected by abstraction of Cα-
proton of the external aldimine and protonation of
C4'-atom of the cofactor. In the reaction of MGL
with its natural substrate such transformation is
one of necessary catalytic stages (Scheme 1) (17).
It seems possible also that the structures III, IIIa,
IV, and V exist in a rapid equilibrium with each
other. Alternatively complex (E•Gly)2 may have a
structure of the enolimine tautomer of the external
aldimine (Scheme 2). Formation of such structure
from the cationic external aldimine (IIIa) may be
possible due to a conformational change of the
protein, associated with an increase of
hydrophobicity of the active site (37,38). Besides a
conformer of the external aldimine with aldimine
bond out of the plane of the cofactor and neutral
form of the enolimine absorb in this region. This
interpretation of the absorbance changes during
binding of glycine is in agreement with the
occurrence of fast Cα-proton exchange. According
to crystal structure of the external aldimine of
MGL with glycine (36) and known role of active
site Lys residue as abstracting pro-(R)-proton in
external aldimines of many PLP-dependent
enzymes (E•Gly)1 conformation fits with that
having orthogonal pro-(R)-proton.
The time courses of absorbance at two single-
wavelengths for the interaction of MGL with
glycine are presented in Figure 1. Single-
wavelength kinetic curves at 420 nm indicate that
the reaction of glycine with MGL consists of two
phases: fast (within a time interval of 20 ms) and
slow (up to 100 s) increase of absorbance (Fig.
1A). On the other hand the absorbance at 320 nm
is characterized by a fast increase for about 100
ms and a slow decrease till 100 s (Fig. 1B). The
absorbance at 500 nm was stable over 200 s
indicating very small level of formation of the
quinonoid intermediate, which evidently is not the
major intermediate.
During the global fitting of kinetic data a
variety of kinetic models were examined, in order
to ascertain the simplest kinetic mechanism that
accurately described the experimental data at 420
nm and 320 nm simultaneously. Global fitting
analysis showed that a reaction mechanism
consisting of three steps and presented in Scheme
3 is the minimal one to appropriately describe the
kinetic data. It should be noted that residuals
(Supplementary Fig. S2) for both 320 nm and 420
nm have the same amplitude of deviation from the
zero line in the whole range of time that gives us
confidence on the reliability of the proposed
kinetic mechanism. The rate and the equilibrium
constants calculated using Scheme 3 are listed in
Table 2. The steady state rate constant (kex) of the
isotopic exchange of the two enantiotopic protons
of glycine (35) are also presented in Table 2.
Having compared the exchange rates with the
other rates in Table 2 it was supposed that the
complex (E•Gly)1, which absorbs at 420 nm, and
is formed in a millisecond time interval, probably
corresponds to the external aldimine intermediate
with orthogonal orientation of the “right” pro-(R)-
proton (Hr) to the cofactor plane (IIIa). Such
conformation is suitable for the exchange of
Reactions of methionine γ-lyase with L-alanine
and L-norvaline – The kinetic traces are similar
for L-alanine and L-norvaline (Fig. 3, 4). In both
cases of interaction of MGL the latest possible
intermediate is an enamine (See Supplementary
Scheme S1). For these amino acids the existence
of exchange of both Сα- and Сβ-protons was
shown (19). Two discernable steps can be noticed
on the absorbance traces. The first one is the
4

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
absorbance decay at 420 nm and the increase at
mixed type (Fig. 5A). It was shown that
application of Dixon plots in conjunction with
double reciprocal plots is useful for the
identification of inhibition behavior (39). The
experimental data were fitted to straight lines in
Dixon plot (Fig. 5B) thus confirming mixed type
of MGL inhibition by L-cycloserine. We were not
able to observe any significant isotopic exchange
of cycloserine protons, at least in conditions
analogous to that in which the exchange had been
observed with other inhibitors.
320 nm (up to 1 s), and the second one is a slow
increase at 420 nm and slow decrease at 320 nm
(up to 1000 s). The absorbance at 500 nm during
interaction MGL with L-alanine and L-norvaline is
slightly changed (data not shown) indicating that
the quinonoid is a temporary intermediate and it is
not accumulated in the reaction. The simplest
mechanism that can account for the kinetic data is
presented in Scheme 4, and includes the
conversion of internal aldimine, absorbing at 420
nm, into complex (E•L-AA)1, which absorbs at
320 nm, and slow conversion of this complex into
the external aldimine (E•L-AA)2, which absorbs at
420 nm. The calculated rate constants of the
formation of the (E•L-AA)2 complex in the
reactions with L-alanine and L-norvaline are much
less than the rate constants of the isotopic
exchange of Cα- and Cβ-protons in the both amino
acids (Table 2). Consequently, the external
aldimine (E•L-AA)2 cannot be an intermediate in
the exchange reactions. It seems likely that in its
structure (IIIc, Fig. 2) the side chain of the bound
amino acid, but not the Cα-proton, is oriented
orthogonally to the PLP, and the exchange of the
Cα-proton is not possible. This structure formally
is analogous to that of the slowly forming external
aldimine (IIIb, Fig. 2) in the reaction with glycine,
responsible for the exchange of the “wrong” pro-
(S)-proton. Thus the isotopic exchange of both
Cα- and Cβ-protons of L-alanine and L-norvaline
should proceed through the intermediate complex
(E•L-AA)1, absorbing at 320 nm. For the
exchange of β-protons the formation of ketimine is
strongly required (17), and we can reasonably
suppose that the complex (E•L-AA)1 corresponds
to the ketimine, existing in a fast equilibrium with
the enolimine tautomer of the external aldimine
also absorbing at 320 nm.
X-ray structure of L-cycloserine–PLP-
derivative
–
Overall conformation of the
polypeptide chains of MGL holoenzyme (PDB ID
2RFV) and MGL-cycloserine complex proved to
be almost identical (RMSD for Cα-atoms of
holoenzyme and the complex is 0.28 Å). There is
the difference in the positions of two protein
loops, N-terminal domain loop 45-60 and C-
terminal domain loop 350-370, located nearby the
active site. In the structures of the holoenzyme
(PDB ID 2RFV and 1Y4I) the N-terminal loop is
notably flexible. In the structure under
consideration, in the structures of Michaelis
complexes of MGL with a number of amino acids
(20), and in the structure of external aldimine with
glycine (36), it is stabilized. Most likely, the
locking of the N-terminal domain loop provides
optimal positioning of two cofactor-binding
residues (Tyr58 and Arg60) and several active site
hydrophobic pocket residues (Phe49, Leu57 and
Leu61). At the same time in spatial structures of
the complexes of the enzyme with amino acids,
the C-terminal segment becomes more flexible. In
the presented structure average B-factor of
residues 350-370 (around 60 Å2) increases as
compared with average B-factor of the same
residues (about 45 Å2) in the holoenzyme structure
(PDB ID 2RV). This flexibility may assist in a
withdrawal of the products from the active site.
We speculate that the N-terminal and the C-
terminal segments make a “shutter” to ensure an
access of a substrate to the active site, efficient
catalysis, and a withdrawal of the products from
the active site.
Inhibition of γ-elimination reaction by L-
cycloserine – Cycloserine is a cyclic analogue of
serine and D-cycloserine is known as natural
antibiotic. L- and D-enantiomers of cycloserine
are inhibitors of a number of PLP-dependent
enzymes belonging to different classes. The γ-
eliminating activity of MGL was reduced in the
course of incubation with L-cycloserine.
Inactivation by L-cycloserine was completely
reversed by dialysis against potassium phosphate
buffer (pH 8.0), containing 0.5 mM PLP and 5
mM DTT. Ki value determined by EnzFitter was
0.068 mM. Linearization of the data in
Lineweaver and Burk plot indicated inhibition of
The resulting 2Fo-Fc and Fo-Fc maps
indicated the presence of cycloserine molecule
covalently bound to PLP (Fig. 6). A well defined
5

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
region of electron density is connected to the
The coenzyme part of the complex is held in
place by all the usual interactions observed in
PLP-dependent enzymes. The phosphate is
anchored by a number of polar interactions with
the protein (14). The N1 atom of the pyridine ring
forms H-bond with carboxylate of Asp185. O3'
hydroxyl of PLP has no contacts with the protein
atoms and is involved in hydrogen bonding with
ketimine N atom of the complex (Fig. 7). In the
structure there are tilts of PLP ring around the C5-
C2 axis and Tyr113 ring to the solvent in
comparison with their positions in the holoenzyme
(25° and 19° respectively). Similar tilts of the
cofactor (17°) and Tyr113 (28°) rings was
demonstrated in three dimensional structure of
external aldimine of the enzyme with glycine (36).
Natom of Lys210 occupies two a little different
positions close to ketimine N atom (~3.5Å). The
abstraction of a Сβ-proton from the first ketimine
intermediate is the next step of γ-elimination
reaction. Data on crystal structure of C. freundii
MGL external aldimine with glycine (36) allowed
us to suppose that in the course of γ-elimination
reaction 1,3-prototropic shift of Cα-proton to C4'-
atom of the cofactor and the abstraction of a β-
proton may proceed with the participation of
Lys210. In the structure under consideration close
positive charge of the ketimine N atom may
stabilize the basic form of Lys210, which allows
the stage of Сβ-proton abstraction to proceed. In
both positions N atom of Lys210 is close (3.75
Å, 3.15 Å) to Cβ-atom of cycloserine ring.
In 1959 several mechanisms were proposed for
interaction of cycloserine with PLP-dependent
enzymes including formation of stable isoxazole
system and an opening of cycloserine ring with
further transformations (42). Evidences for first
mechanism were obtained in 1998 for γ-
aminobutyric acid aminotransferase (43) and for
D-amino acid aminotransferase (44). It was
proposed that an abstraction of a β-proton of
cycloserine from ketimine by active site lysine
residue leads to stable aromatic cycloserine-PLP
complex. At the resolution of our structure we do
not exclude a saturation of N-CA bond but the
geometry of the complex is more compatible with
double N-CA bond. For mechanisms of γ-
elimination and γ-replacement reactions catalyzed
by PLP-dependent enzymes it was postulated that
ketimine (Scheme 1, V) is plausible intermediate
for effective labilization of Cβ-proton to proceed
(17). Its formation in the course of these reactions
catalyzed by E. coli cystathionine γ-synthase was
cofactor and is clearly separated from any protein
residues, including N atom of Lys210, which
forms covalent bond with PLP in the holoenzyme.
We attempted to incorporate two tautomers of
PLP-bound L-cycloserine into the electron density.
One of them should possess planarity of the five-
membered ring due to presence of double bond
between C and N atoms of the cycloserine; the
second does not have this restrain and could be
bent. Comparison of the possible fitting of the two
structures revealed better matching of the planar
cycloserine to the experimental electron density.
The C3-C4-C4A-N dihedral angle is -5.1° and
constrained by hydrogen bond between N and O3’
atoms. The C4-C4A-N-CA dihedral angle is
167.4° and the C4A-N-CA-CB dihedral angle is -
4.7° so the plane of the L-cycloserine is somewhat
tilted with respect to the pyridine ring (Fig. 6). The
geometry of the complex is corresponding to sp3
hybridization of C4A which is implied by the shift
of the band of MGL internal aldimine at 420 nm to
320 nm observed in polarized absorption spectra
of holoenzyme crystal in the presence of L-
cycloserine (40) and in absorption spectrum of the
complex in
a
solution (data not shown).
Accordingly, the N-CA bond of the complex
should be double; the exocyclic oxygen atom
connects to the cycloserine ring by single bond
and is protonated. The structure corresponds to
ketimine intermediate (Scheme 5) and matches
well with that described for the complex of
dialkylglycine decarboxylase with L-cycloserine
(41).
The stereochemical position of the L-
cycloserine-PLP complex in the active site is
determined by firmly fixed contacts with amino
acid residues of two subunits which are involved
in the organization of a catalytic dimer. The
endocyclic oxygen of the ring interacts with the
hydrogen of NH group of main chain Ser339 from
one side and with nearest water molecule from the
other side (Fig. 7). The exocyclic oxygen makes
hydrogen bonds with NH1 and NH2 atoms of
Arg374. This residue is involved in a binding of
carboxylic groups in the external aldimine of
MGL with glycine (36) and in Michaelis
complexes of the enzyme with some substrates
and inhibitors (20). The position of the bound
cycloserine is additionally stabilized by stacking
with side chains of Tyr58 from the neighboring
subunit and with Tyr113 of its own protein chain.
6

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
detected by stopped-flow data in the same paper.
bindings by MGL showed that the equilibrium
constant of the first step K1 = k1/k-1 is increased in
the row: glycine < L-norvaline ~ L-alanine < L-
cycloserine. The rate constant of the forward
reaction k1 for glycine, L-norvaline and L-
cycloserine is in the range of (1.0-4.0)×104 M-1s-1,
being at least 10 times higher than in the case of
L-alanine (Table 2). Interestingly, the rate constant
of the isotopic exchange of Cα-protons for L-
alanine is 10 times smaller than that of glycine and
L-norvaline (19). The process characterized with
the rate constant k1 should represent the complex
mechanism including not only multistage process
of the Schiff base formation between incoming
amino acid and Lys210, but possibly the enzyme
reorganization during the incorporation of the
amino acid into the active site. The rate constant k-
1, which characterizes the decomposition of the
first complex (Table 2), reflects the stability of this
state. In cases of L-alanine and L-cycloserine the
magnitude of k-1 is more than 150 and 50 times
lower than the same value for glycine and L-
norvaline.
Formation of the “right” external aldimine
enables the formation of the ketimine intermediate
which was obviously registered in the cases of L-
norvaline and L-alanine and probably for glycine
([E•L-Nva]1, [E•L-Ala]1 or ([E•Gly]2 complexes,
respectively). The last detected complex (([E•L-
Nva]2, [E•L-Ala]2 or ([E•Gly]3) likely
corresponds to the external aldimine in the
“wrong” conformation. In the case of L-
cycloserine the formation of (E•L-cSer)1 complex
reflects the formation of the external aldimine. The
subsequent transformations (abstraction of Cα-
proton and protonation of C4'-atom of PLP) lead
to accumulation of stable ketimine intermediate.
The kinetic data have shown that amino acids
incorporate in the active site of MGL in the proper
conformation owing to the conformational
selectivity of the active site. The Schiff bases can
be formed with an amino acid only having “right”
orientation of the side chain, Cα-hydrogen and
carboxyl group relatively to PLP plane. After the
formation of the ketimine intermediate the flat
structure of amino acid can be turned in the active
site of enzyme thus after the reversible protonation
the amino acid would be in the “wrong”
conformation (e.g. IIIb, Fig. 2).
The evidence of ketimine intermediate was
obtained by X-ray data for cystathionine γ-
synthase (45).
Reaction of methionine γ-lyase with L-
cycloserine – In steady-state absorption spectrum
of MGL complexed with L-cycloserine the band
belonging to the internal aldimine with maximum
in the region 420 nm disappeared along with the
appearance the predominant band at the region
320-330 nm (data not shown).
The reaction of MGL with L-cycloserine leads
to a single change of absorbance within 2000 s: a
decrease at 420 nm and an increase at 320 nm, the
rates of the two processes being identical (Fig. 8).
According to X-ray data the possible intermediate
of the reaction of MGL with L-cycloserine is
ketimine, which has absorbance maximum at the
region of 320 nm. It can be assumed that the
observed spectral changes correspond to the
decomposition of external aldimine and
accumulation of ketimine, respectively (Scheme
5). The external aldimine is converted to a
ketimine intermediate without detectable changes
at 500 nm, indicating that the intermediate
quinonoid species are not accumulating in the
reaction.
The processing of the L-cycloserine by MGL
was described by kinetic Scheme 4. The values for
individual kinetic constants were determined by
global fitting (Table 2). (E•L-AA)1 complex more
likely corresponds to external aldimine, which
subsequently transforms in ketimine in the
complex (E•L-AA)2 (Scheme 5). Interestingly, the
forward rate constant k1 and k2 are similar to the
respective constants for L-norvaline reaction. At
the same time the reverse rate constant k-1 and k-2
are 58 and 1.6 times less as compared to L-
norvaline reaction, indicating that MGL
complexes with L-cycloserine are more stable,
which leads to shift of the equilibrium to the
ketimine intermediate.
The stability of ketimine intermediate may
explain the higher inhibition of the reaction with
L-methionine in comparison with glycine, L-
alanine and L-norvaline. The Ki value of inhibition
of γ-elimination of L-methionine by L-cycloserine
is at least tenfold less that the Ki values for
glycine, L-alanine and L-norvaline (Table 2).
In general, both X-ray and kinetic analyses of
the interaction of MGL with L-cycloserine provide
the direct proof of ketimine intermediate formation
CONCLUSION
The comparison of values of the rate constants
characterizing the processes of the amino acids
7

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
in the catalytic mechanism of PLP-dependent γ-
elimination reaction.
8

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
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Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
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Stereospecificity of isotopic exchange of C-alpha-protons of glycine catalyzed by three PLP-
dependent lyases: the unusual case of tyrosine phenol-lyase. Amino acids 41, 1247-1256
Revtovich, S. V., Faleev, N. G., Morozova, E. A., Anufrieva, N. V., Nikulin, A. D., and Demidkina, T.
V. (2014) Crystal structure of the external aldimine of Citrobacter freundii methionine gamma-
lyase with glycine provides insight in mechanisms of two stages of physiological reaction and
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Johnson, G. F., Tu, J. I., Bartlett, M. L., and Graves, D. J. (1970) Physical-chemical studies on the
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Shaltiel, S., and Cortijo, M. (1970) The mode of binding of pyridoxal 5'-phosphate in glycogen
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Butterworth, P. J. (1972) The use of Dixon plots to study enzyme inhibition. Biochim. Biophys.
Acta 289, 251-253
Ronda, L., Bazhulina, N. P., Morozova, E. A., Revtovich, S. V., Chekhov, V. O., Nikulin, A. D.,
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Kochetkov, N. K., Khomutov, R. M., Karpeisky, M. Y., Bydovsky, E. I., and Severin, E. S. (1959) The
mechanism of antibiotic action of cycloserine. Doklady Akademii Nayk SSSR 129, 1132-1134
Olson, G. T., Fu, M. M., Lau, S., Rinehart, K. L., and Silverman, R. B. (1998) An aromatization
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Peisach, D., Chipman, D. M., Van Ophem, P. W., Manning, J. M., and Ringe, D. (1998) D-
cycloserine inactivation of D-amino acid aminotransferase leads to a stable noncovalent protein
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Steegborn, C., Laber, B., Messerschmidt, A., Huber, R., and Clausen, T. (2001) Crystal structures
of cystathionine gamma-synthase inhibitor complexes rationalize the increased affinity of a
novel inhibitor. J. Mol. Biol. 311, 789-801
11

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
FOOTNOTES
*This work was supported by the Program of the Russian Academy of Sciences “Molecular & Cell
Biology” (6.11); Russian Foundation for Basic Researches (14-04-00349), the Grant from Russian
Scientific Foundation (14-14-00063), the grants of the President of the Russian Federation for state
support of the leading scientific schools (SS-2064.2014.4, SS-1205.2014.4), Russian Ministry of
Education and Science (SP-4012.2013.4).
§To whom correspondence should be addressed. T.V.D. Tel.: +7(499)135 9858, Email: tvd@eimb.ru;
O.S.F. Tel: +7(383)363 5174; Email: fedorova@niboch.nsc.ru
1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of
Sciences, Novosibirsk 630090, Russia
2Department of Natural Sciences, Novosibirsk State University, Novosibirsk 630090, Russia
3Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Moscow 119991,
Russia
4Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow 119991, Russia
5Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142290,
Russia
6The abbreviations used are: MGL, methionine γ-lyase; PLP, pyridoxal 5'-phosphate; L-cSer, L-
cycloserine; L-Nva, L-norvaline; PDB, Protein Data Bank.
12

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
SCHEMES AND FIGURES LEGENDS
Scheme 1. Chemical mechanism of the γ-elimination reaction.
Scheme 2. The possible intermediates in the reaction of glycine with MGL.
Scheme 3. Kinetic mechanism of MGL interaction with glycine.
Scheme 4. Kinetic mechanism of MGL processing of L-alanine, L-norvaline and L-cycloserine.
Scheme 5. The possible intermediates in the reaction of L-cycloserine with MGL.
FIGURE 1. Interaction of MGL and glycine as monitored by changes of absorbance of the
holoenzyme. Time courses were recorded at pH 7.8, 0.1 M potassium phosphate buffer solution,
containing 0.5 mM DTT and 0.1 mM EDTA, with the concentration of MGL fixed at 12.5 µM. Time
courses were obtained at 25 °C. Solution of enzyme was mixed with various concentrations of glycine
(10-500 mM). Each kinetic curve was averaged over at least four independent experiments. The
absorbance at 420 nm (A) and 320 nm (B) was detected. Solid lines represent the fitted curves.
FIGURE 2. Schematic representation of possible aldimine conformers of amino acids in the active
site of MGL.
FIGURE 3. Interaction of MGL and L-alanine as monitored by changes of absorbance of the
holoenzyme. Time courses were recorded at pH 7.8, 0.1 M potassium phosphate buffer solution,
containing 0.5 mM DTT and 0.1 mM EDTA, with the concentration of MGL fixed at 12.5 µM. Time
courses were obtained at 25 °C. Solution of enzyme was mixed with various concentrations of L-alanine
(1.0-12.0 mM). Each kinetic curve was averaged over at least four independent experiments. The
absorbance at 420 nm (A) and 320 nm (B) was detected. Solid lines represent the fitted curves.
FIGURE 4. Interaction of MGL and L-norvaline as monitored by changes of absorbance of the
holoenzyme. Time courses were recorded at pH 7.8, 0.1 M potassium phosphate buffer solution,
containing 0.5 mM DTT and 0.1 mM EDTA, with the concentration of MGL fixed at 12.5 µM. Time
courses were obtained at 25 °C. Solution of enzyme was mixed with various concentrations of L-
norvaline (1.5-7.5 mM). Each kinetic curve was averaged over at least four independent experiments. The
absorbance at 420 nm (A) and 320 nm (B) was detected. Solid lines represent the fitted curves.
FIGURE 5. The inhibition of the -elimination reaction by L-cycloserine. Lineweaver and Burk (A)
and Dixon (B) plots.
FIGURE 6. Stereo view of PLP-cycloserine derivative with the final 2Fo – Fc map contoured at the
1.5 σ level.
FIGURE 7. Stereo view of the active site. H-bonds are indicated by green dashed lines. The shown
distances are in Å.
FIGURE 8. Interaction of MGL and L-cycloserine as monitored by changes of absorbance of the
holoenzyme. Time courses were recorded at pH 7.8, 0.1 M potassium phosphate buffer solution,
containing 0.5 mM DTT and 0.1 mM EDTA, with the concentration of MGL fixed at 12.5 µM. Time
courses were obtained at 25 °C. Solution of enzyme was mixed with various concentrations of L-
cycloserine (6.35-38.1 µM). Each kinetic curve was averaged over at least three independent experiments.
The absorbance at 420 nm (A) and 320 nm (B) was detected. Solid lines represent the fitted curves.
13

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
TABLES
Table 1. Data collection and refinement statistics. Values in parentheses are for the highest resolution
shell.
Space group
I222
Unit cell parameters (Å)
a = 56.27, b = 122.89, c = 126.61,
α=β=γ=90
0.843
15-1.60 (1.65-1.60)
55080
17.3 (3.6)
94.5 (94.3)
4.5 (3.9)
Wavelength (Å)
Resolution (Å)
No. of unique reflections
Mean I/σ(I)
Completeness (%)
Redundancy
Rsym (%)
4.8 (39.6)
Refinement
Resolution range (Å)
Rwork
Rfree
No. of protein atoms
No. of water atoms
No. of heterogenic atoms
15-1.6 (1.64-1.60)
0.139 (0.197)
0.275 (0.293)
2993
266
57
Mean temperature factor B 28.69
(Å2)
R.m.s. deviation from ideal
values
Bond lengths (Å)
Bond angles (°)
0.019
1.985
Ramachandran plot
Favoured region (%)
Allowed region (%)
Outlier region (%)
97.11
2.10
0.79
14

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
Table 2. Pre-steady-state and isotopic exchange kinetic parameters for interaction of MGL (from
Citrobacter freundii) with amino acids.
Constants*
k1, M-1s-1
k-1, s-1
k2, s-1
Glycine
(1.0 ± 0.2)×104
140 ± 30
1.5 ± 0.1
33.5 ± 1.3
0.3 ± 0.1
0.08 ± 0.04
14
L-alanine
(7.0 ± 0.1)×102
0.6 ± 0.1
L-norvaline
(4.0 ± 1.0)×104
94.0 ± 6.0
L-cycloserine
(3.6 ± 1.2)×104
1.6 ± 0.3
(6.5 ± 2.5)×10-3
(3.8 ± 0.4)×10-4
(6.8 ± 0.5)×10-3 (6.6 ± 0.4)×10-3
k-2, s-1
k3, s-1
(3.6 ± 0.4)×10-3 (6.3 ± 0.2)×10-3
-
-
-
2.3
1.0
k-3, s-1
K1, mM
K2
-
0.045
0.58
0.9
0.53
22.3
K3
0.27
Ki, mM
49**
0.068±0.07
3.4**
2.71
2.63
4.7**
20.6
11.1
kexα-H, s-1 ***
20.2 pro-(R)
-
1.4×10-3 pro-(S)
kexβ-H, s-1 ***
* Kn = k-n/kn
-
-
** Ki for glycine, L-alanine and L-norvaline from (19).
*** kex for glycine from (35), L-alanine and L-norvaline from (19).
15

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
Scheme 1. (2-column width)
16

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
Scheme 2. (2-column width)
17

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
Scheme 3. (1.5-column width)
18

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
Scheme 4. (1-column width)
19

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
Scheme 5. (2-column width)
20

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
Figure 1.
(1-column width)
21

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
Figure 2.
(1-column width)
22

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
Figure 3.
(1-column width)
23

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
Figure 4.
(1-column width)
24

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
Figure 5.
(1-column width)
25

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
Figure 6.
(1.5-column width)
26

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
Figure 7.
(2-column width)
27

Kinetic and Structural Studies of Inhibitors for Methionine γ-Lyase
Figure 8.
(1-column width)
28