Properties of intramolecular proton transfer in carbonic anhydrase III.

We investigated the efficiency of glutamic acid 64 and aspartic acid 64 as proton donors to the zinc-bound hydroxide in a series of site-specific mutants of human carbonic anhydrase III (HCA III). Rate constants for this intramolecular proton transfer, a step in the catalyzed dehydration of bicarbonate, were determined from the proton-transfer-dependent rates of release of H2 18O from the enzyme measured by mass spectrometry. The free energy plots representing these rate constants could be fit by the Marcus rate theory, resulting in an intrinsic barrier for the proton transfer of deltaG0++ = 2.2 +/- 0.5 kcal/mol, and a work function or thermodynamic contribution to the free energy of reaction wr = 10.8 +/- 0.1 kcal/mol. These values are very similar in magnitude to the Marcus parameters describing intramolecular proton transfer from His64 and His67 to the zinc-bound hydroxide in mutants of HCA III. That result and the equivalent efficiency of Glu64 and Asp64 as proton donors in the catalysis by CA III demonstrate a lack of specificity in proton transfer from these sites, which is indirect evidence of a number of proton conduction pathways through different structures of intervening water chains. The dominance of the thermodynamic contribution or work function for all of these proton transfers is consistent with the view that formation and breaking of hydrogen bonds in such water chains is a limiting factor for proton translocation.     

Catalytic mechanism of aromatic prenylation by NphB

NphB is an aromatic prenyltransferase that catalyzes the attachment of a 10-carbon geranyl group to aromatic substrates. Importantly, NphB exhibits a rich substrate selectivity and product regioselectivity. A systematic computational study has been conducted in order to address several question associated with NphB-catalyzed geranylation. The reaction mechanism of the prenylation step has been characterized as a S(N)1 type dissociative mechanism with a weakly stable carbocation intermediate. A novel π-chamber composed of Tyr121, Tyr216, and 1,6-DHN is found to be important in stabilizing the carbocation. The observed difference in the rates of product formation from 5- and 2-prenylation arises from the differing orientations of the aromatic substrate in the resting state. 4-Prenylation shares the same resting state with 5-prenylation, but the lower free energy barrier for carbocation formation makes the latter reaction more facile. The high free energy barrier associated with 7-prenylation is caused by the unfavorable orientation of 1,6-DHN in active site pocket, along with the difficulty of proton elimination after the prenylation step. A water-mediated proton transfer facilitates the loss of hydrogen at the prenylation site to form the final prenylated product. Interestingly, the same crystallographically observed water molecule has been found to be responsible for proton loss in all three experimentally identified products. After proton transfer, the relaxation of the final product from a sp(3) carbon center to a sp(2) center triggers a "spring-loaded" product release mechanism which pushes the final product out of the binding pocket toward the edge of the active site. The hydrogen bond interactions between the two hydroxyl groups of the aromatic product and the side chains of Ser214 and Tyr288 help to "steer" the movement of the product. In addition, mutagenesis studies identify these same two side chains as being responsible for the observed regioselectivity, particularly 2-prenylation. These observations provide valuable insights into NphB chemistry, offering an opportunity to better engineer the active site and to control the reactivity in order to obtain high yields of the desired product(s). Furthermore, the S(N)1 reaction mechanism observed for NphB differs from the prenylation reaction found in, for example, the farnesyltransferase, which proceeds via an S(N)2-like reaction pathway. The spring-loaded release mechanism highlighted herein also offers novel insights into how enzymes facilitate product release.     

Oxygen reactions in p-hydroxybenzoate hydroxylase utilize the H-bond network during catalysis

para-Hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyses a reaction in two parts: reduction of the flavin adenine dinucleotide (FAD) in the enzyme by reduced nicotinamide adenine dinucleotide phosphate (NADPH) in response to binding p-hydroxybenzoate to the enzyme and oxidation of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates p-hydroxybenzoate. These different reactions are coordinated through conformational rearrangements of the protein and isoalloxazine ring during catalysis. Earlier research showed that reduction of FAD occurs when the isoalloxazine of the FAD moves to the surface of the protein to allow hydride transfer from NADPH. This move is coordinated with protein rearrangements that are triggered by deprotonation of buried p-hydroxybenzoate through a H-bond network that leads to the surface of the protein. In this paper, we examine the involvement of this same H-bond network in the oxygen reactions-the initial formation of a flavin-C4a-hydroperoxide from the reaction between oxygen and reduced flavin, the electrophilic attack of the hydroperoxide upon the substrate to form product, and the elimination of water from the flavin-C4a-hydroxide to form oxidized enzyme in association with product release. These reactions were measured through absorbance and fluorescence changes in the FAD during the reactions. Results were collected over a range of pH for the reactions of wild-type enzyme and a series of mutant enzymes with the natural substrate and substrate analogues. We discovered that the rate of formation of the flavin hydroperoxide is not influenced by pH change, which indicates that the proton required for this reaction does not come from the H-bond network. The rate of the hydroxylation reaction increases with pH in a manner consistent with a pK(a) of 7.1. We conclude that the H-bond network abstracts the phenolic proton from p-hydroxybenzoate in the transition state of oxygen transfer. The rate of formation of oxidized enzyme increases with pH in a manner consistent with a pK(a) of 7.1, indicating the involvement of the H-bond network. We conclude that product deprotonation enhances the rate of a specific conformational change required for both product release and the elimination of water from C4a-OH-FAD.     

Dissecting the pattern of proton release from partial process involved in ubihydroquinone oxidation in the Q-cycle

A key feature of the modified Q-cycle of the cytochrome bc₁ and related complexes is a bifurcation of QH₂ oxidation involving electron transfer to two different acceptor chains, each coupled to proton release. We have studied the kinetics of proton release in chromatophore vesicles from Rhodobacter sphaeroides, using the pH-sensitive dye neutral red to follow pH changes inside on activation of the photosynthetic chain, focusing on the bifurcated reaction, in which 4H⁺are released on complete turnover of the Q-cycle (2H⁺/ubiquinol (QH₂) oxidized). We identified different partial processes of the Q_o-site reaction, isolated through use of specific inhibitors, and correlated proton release with electron transfer processes by spectrophotometric measurement of cytochromes or electrochromic response. In the presence of myxothiazol or azoxystrobin, the proton release observed reflected oxidation of the Rieske iron?sulfur protein. In the absence of Q_o-site inhibitors, the pH change measured represented the convolution of this proton release with release of protons on turnover of the Q_o-site, involving formation of the ES-complex and oxidation of the semiquinone intermediate. Turnover also regenerated the reduced iron-sulfur protein, available for further oxidation on a second turnover. Proton release was well-matched with the rate limiting step on oxidation of QH₂ on both turnovers. However, a minor lag in proton release found at pH?7 but not at pH?8 might suggest that a process linked to rapid proton release on oxidation of the intermediate semiquinone involves a group with a pK in that range.     

Effect of ammonia on the glutamate dehydrogenase catalyzed oxidative deamination of L-glutamate. The steady state

Ammonia is known to inhibit the steady-state rate of oxidation of L-glutamate catalyzed by glutamate dehydrogenase. We reported previously [Brown, A., Colen, A. H., & Fisher, H. F. (1978) Biochemistry 17, 2031] kinetic evidence supporting the formation in the initial rapid phase of a complex which is composed of enzyme, reduced coenzyme, alpha-ketoglutarate, and ammonia. We show here that the effects of ammonia on the steady-state reaction can be correlated with transient-state kinetic effects related to the concentration of that ammonia-containing complex. These results indicate the existence of alternate reaction pathways which become important at high ammonia concentrations. These new pathways provide an additional route for the release of NADPH from the enzyme surface. The expanded mechanism shows that the noncompetitive product inhibition by ammonia can occur without the simultaneous presence of ammonia and L-glutamate on the enzyme. This mechanism also accommodates the observed substrate inhibition by L-glutamate.     

Short-strong hydrogen bonds and a low barrier transition state for the proton transfer reaction in RNase A catalysis: a quantum chemical study

There is growing evidence that some enzymes catalyze reactions through the formation of short-strong hydrogen bonds as first suggested by Gerlt and Gassman. Support comes from several experimental and quantum chemical studies that include correlation energies on model systems. In the present study, the process of proton transfer between hydroxyl and imidazole groups, a model of the crucial step in the hydrolysis of RNA by the enzymes of the RNase A family, is investigated at the quantum mechanical level of density functional theory and perturbation theory at the MP2 level. The model focuses on the nature of the formation of a complex between the important residues of the protein and the hydroxyl group of the substrate. We have also investigated different configurations of the ground state that are important in the proton transfer reaction. The nature of bonding between the catalytic unit of the enzyme and the substrate in the model is investigated by Bader's atoms in molecule theory. The contributions of solvation and vibrational energies corresponding to the reactant, the transition state and the product configurations are also evaluated. Furthermore, the effect of protein environment is investigated by considering the catalytic unit surrounded by complete proteins--RNase A and Angiogenin. The results, in general, indicate the formation of a short-strong hydrogen bond and the formation of a low barrier transition state for the proton transfer model of the enzyme.     

Proton transfer facilitated by ligand binding. An energetic analysis of the catalytic mechanism of Trypanosoma cruzi trans-sialidase

Trans-sialidase is a crucial enzyme for the infection of Trypanosoma cruzi, the protozoa responsible for Chagas' disease in humans. This enzyme catalyzes the transfer of sialic acids from mammalian host cells to parasitic cell surfaces in order to mask the infection from the host's immune system. It represents a promising target for the development of therapeutics to treat the disease and has been subject of extensive structural studies. Elaborate experiments suggested formation of a long-lived covalent intermediate in the catalytic mechanism and identified a Tyr/Glu pair as an unusual catalytic couple. This requires that the tyrosine hydroxyl proton is transferred to the carboxylate group of glutamate before the nucleophilic attack. Since the solution pK(a)s of tyrosine and glutamate are very different, this transfer can only be accomplished if the reaction environment selectively stabilizes the product state. We compute the free energy profile for the proton transfer in different environments, and our results indicate that it can take place in the active site of trans-sialidase, but only after substrate binding. By means of the energy decomposition method, we explain the influence that the active site residues exert on the reaction and how the pattern is changed when the substrate is present. This study represents an initial step that can shed light on our understanding of the catalytic mechanism of this reaction.     

Retardation of proton transfer caused by binding of the transition metal ion to the bacterial reaction center is due to pKa shifts of key protonatable residues

Transition metal ions bind to the reaction center (RC) protein of the photosynthetic bacterium Rhodobacter sphaeroides and slow the light-induced electron and proton transfer to the secondary quinone, Q(B). We studied the properties of the metal ion-RC complex by measuring the pH dependence of the dissociation constant and the stoichiometry of proton release upon ligand formation. We investigated the mechanism of inhibition by measuring the stoichiometry and kinetics of flash-induced proton binding, the transfer of (first and second) electrons to Q(B), and the rate of steady-state turnover of the RC in the absence and presence of Cd(2+) and Ni(2+) on a wide pH range. The following results were obtained. (1) The complexation of transition metal ions Cd(2+) and Ni(2+) with the bacterial RC showed strong pH dependence. This observation was explained by different (pH-dependent) states of the metal-ligand cluster: the complex formation was strong when the ligand (Asp and His residues) was deprotonated and was much weaker if the ligand was partly (or fully) protonated. A direct consequence of the model was the pH-dependent proton release upon complexation. (2) The retardation of transfer of electrons and protons to Q(B) was also strongly pH-dependent. The effect was large in the neutral pH range and decreased toward the acidic and alkaline pH values. (3) Steady-state turnover measurements indicated that the rate of the second proton transfer was much less inhibited than that of the first one, which became the rate-limiting step in continuous turnover of the RC. (4) Sodium azide partly recovered the proton transfer rate. The effect is not due to removal of the bound metal ion by azide but probably by formation of a proton-transporting azide network similarly as water molecules may build up proton pathways. (5) We argue that the inhibition comes mainly from pK(a) shifts of key protonatable residues that control the proton transfer along the H-bond network to Q(B). The electrostatic interaction between the metal ion and these residues may result in acidic pK(a) shifts between 1.5 and 2.0 that account for the observed retardation of the electron and proton transfer.     

Uridine phosphorylase from Trypanosoma cruzi: kinetic and chemical mechanisms

The reversible phosphorolysis of uridine to generate uracil and ribose 1-phosphate is catalyzed by uridine phosphorylase and is involved in the pyrimidine salvage pathway. We define the reaction mechanism of uridine phosphorylase from Trypanosoma cruzi by steady-state and pre-steady-state kinetics, pH-rate profiles, kinetic isotope effects from uridine, and solvent deuterium isotope effects. Initial rate and product inhibition patterns suggest a steady-state random kinetic mechanism. Pre-steady-state kinetics indicated no rate-limiting step after formation of the enzyme-products ternary complex, as no burst in product formation is observed. The limiting single-turnover rate constant equals the steady-state turnover number; thus, chemistry is partially or fully rate limiting. Kinetic isotope effects with [1'-(3)H]-, [1'-(14)C]-, and [5'-(14)C,1,3-(15)N(2)]uridine gave experimental values of (α-T)(V/K)(uridine) = 1.063, (14)(V/K)(uridine) = 1.069, and (15,β-15)(V/K)(uridine) = 1.018, in agreement with an A(N)D(N) (S(N)2) mechanism where chemistry contributes significantly to the overall rate-limiting step of the reaction. Density functional theory modeling of the reaction in gas phase supports an A(N)D(N) mechanism. Solvent deuterium kinetic isotope effects were unity, indicating that no kinetically significant proton transfer step is involved at the transition state. In this N-ribosyl transferase, proton transfer to neutralize the leaving group is not part of transition state formation, consistent with an enzyme-stabilized anionic uracil as the leaving group. Kinetic analysis as a function of pH indicates one protonated group essential for catalysis and for substrate binding.     

Bovine lenticular gamma-glutamylcysteine synthetase: reaction sequence.

The sequence of substrate addition and product release during the reaction catalyzed by gamma-glutamylcysteine synthetase was investigated with purified enzyme from bovine lens. Thermal inactivation and kinetic studies suggest that L-glutamate is the first substrate to bind to the enzyme. L-beta-Chloroalanine was used as the L-cysteine analogue. Utilizing substrate activation and product inhibition studies, the following reaction sequence was determined: L-glutamate binding. ATP binding, ADP release, L-beta-chloroalanine binding, followed by inorganic phosphate and then dipeptide release. The implications of this mechanism with regard to control of the enzyme in situ and its importance in glutathione synthesis are discussed.     

Local protein dynamics and catalysis: detection of segmental motion associated with rate-limiting product release by a glutathione transferase

Glutathione transferase rGSTM1-1 catalyzes the addition of glutathione (GSH) to 1-chloro-2,4-dinitrobenzene, a reaction in which the chemical step is 60-fold faster than the physical step of product release. The hydroxyl group of Y115, located in the active site access channel, controls the egress of product from the active site. The Y115F mutant enzyme has a k(cat) (72 s(-)(1)) that is 3.6-fold larger than that of the native enzyme (20 s(-)(1)). Crystallographic observations and evidence from amide proton exchange kinetics are consistent with localized increases in the degree of segmental motion of the Y115F mutant that are coupled to the enhanced rate of product release. The loss of hydrogen bonding interactions involving the hydroxyl group of Y115 is reflected in subtle alterations in the backbone position, an increase in B-factors for structural elements that comprise the channel to the active site, and, most dramatically, a loss of well-defined electron density near the site of mutation. The kinetics of amide proton exchange are also enhanced by a factor between 3 and 12 in these regions, providing direct, quantitative evidence for changes in local protein dynamics affecting product release. The enhanced product release rate is proposed to derive from a small shift in the equilibrium population of protein conformers that permit egress of the product from the active site.     

Mechanism of action of Drosophila melanogaster alcohol dehydrogenase.

Reported kinetic pH dependence data for alcohol dehydrogenase from Drosophila melanogaster are analyzed with regard to differences in rate behaviour between this non-metallo enzyme and the zinc-containing liver alcohol dehydrogenase present in vertebrates. For the Drosophila enzyme a mechanism of action is proposed according to which catalytic proton release to solution during alcohol oxidation occurs at the binary-complex level as an obligatory step preceding substrate binding. Such proton release involves an ionizing group with a pKa of about 7.6 in the enzyme.NAD+ complex, tentatively identified as a tyrosyl residue. The ionized form of this group is proposed to participate in the binding of alcohol substrates and to act as a nucleophilic catalyst of the subsequent step of hydride ion transfer from the bound alcohol to NAD+. Herein lie fundamental mechanistic differences between the metallo and non-metallo short chain alcohol dehydrogenases.     

Histidine is involved in coupling proton uptake to electron transfer in photosynthetic proteins

In photosynthesis, the central step in transforming light energy into chemical energy is the coupling of light-induced electron transfer to proton uptake and release. Despite intense investigations of different photosynthetic protein complexes, including the photosystem II (PS II) in plants and the reaction center (RC) in bacteria, the molecular details of this fundamental process remain incompletely understood. In the RC of Rhodobacter (Rb.) sphaeroides, fast formation of the charge separated state, P(+)Q(A)(-), is followed by a slower electron transfer from the primary acceptor, Q(A), to the secondary acceptor, Q(B), and the uptake of a proton from the cytoplasm. The proton transfer to Q(B) takes place via a protonated water chain. Mutation of the amino acid AspL210 to Asn (L210DN mutant) near the entry of the proton pathway can disturb this water chain and consequently slow down proton uptake. Time-resolved step-scan Fourier transform infrared (FTIR) measurements revealed an intermediate X in the Q(A)(-)Q(B) to Q(A)Q(B)(-) transition. The nature of this transition remains a matter of debate. In this study, we investigated the role of the iron-histidine complex located between Q(A) and Q(B). We used time-resolved fast-scan FTIR spectroscopy to characterize the Rb. sphaeroides L210DN RC mutant marked with isotopically labeled histidine. FTIR marker bands of the intermediate X between 1120 cm(-1) and 1050 cm(-1) are assigned to histidine vibrations and indicate the protonation of a histidine, most likely HisL190, during the disappearance of the intermediate. Based on these results we propose a novel mechanism of the coupling of electron and proton transfer in photosynthesis.     

Considerations on the mechanism of photosynthetic water oxidation – dual role of oxo-bridges between Mn ions in (i) redox-potential maintenance and (ii) proton abstraction from substrate water

Two mechanistic problems of photosynthetic water oxidation at the Mn complex of Photosystem II (PS II) are considered. (I) In the four Mn-oxidizing transitions, any pure Mn oxidation is predicted to cause an increase in redox potential that renders subsequent oxidation steps impossible (redox-potential problem). Formation of unprotonated oxo-bridges may counteract the potential increase. (II) The O–O formation step without any high-pK bases acting as proton acceptors is energetically unfavorable (acceptor-base problem). The pK of oxides in a bridging position between Mn ions may increase drastically upon reduction of Mn in the water-oxidation step (>10 units), thus rendering them favorable proton acceptors. It is proposed that in PS II, in the course of the four oxidizing transitions at least two unprotonated oxo-bridges are formed. Thereby (i) a redox potential increase is prevented and (ii) proton acceptors are prepared for the O–O formation step. Water oxidation in the O–O bond formation step is facilitated by simultaneous Mn reduction and proton transfer to bridging oxides amounting to hydrogen atom or hydride transfer from substrate water to the Mn-oxo core of the Mn complex of PS II.     

Catalytic cycle of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus. Solvent viscosity, deuterium isotope effects, and proton inventory studies

The phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLCBc) is a 28.5 kDa enzyme with three zinc ions in its active site. Although much is known about the roles that various PLCBc active site amino acids play in binding and catalysis, there is little information about the rate-determining step of the PLCBc-catalyzed hydrolysis of phospholipids and the catalytic cycle of the enzyme. To gain insight into these aspects of the hydrolysis, solvent viscosity variation experiments were conducted to determine whether an external step (substrate binding or product release) or an internal step (hydrolysis) is rate-limiting. The data indicate that the PLCBc-catalyzed reaction is unaffected by changes in solvent viscosity. This observation is inconsistent with the notion of substrate binding or product release being rate-determining and supports the hypothesis that a chemical step is rate-limiting. Furthermore, a deuterium isotope effect of 1.9 and a linear proton inventory plot indicate one proton is transferred in the rate-determining step. These data may be used to formulate a comprehensive catalytic cycle that is for the first time based on experimental evidence. In this mechanism, Asp55 of PLCBc activates an active site water molecule for attack on the phosphodiester bond, the hydrolysis of which is rate-limiting. The phosphorylcholine product is the first to leave the active site, followed by diacylglycerol.     

Pre-steady-state kinetic investigation of intermediates in the reaction catalyzed by adenosylcobalamin-dependent glutamate mutase.

Glutamate mutase catalyzes the reversible isomerization of L-glutamate to L-threo-3-methylaspartate. Rapid quench experiments have been performed to measure apparent rate constants for several chemical steps in the reaction. The formation of substrate radicals when the enzyme was reacted with either glutamate or methylaspartate was examined by measuring the rate at which 5'-deoxyadenosine was formed, and shown to be sufficiently fast for this step to be kinetically competent. Furthermore, the apparent rate constant for 5'-deoxyadenosine formation was very similar to that measured previously for cleavage of the cobalt-carbon bond of adenosylcobalamin by the enzyme, providing further support for a mechanism in which homolysis of the coenzyme is coupled to hydrogen abstraction from the substrate. The pre-steady-state rates of methylaspartate and glutamate formation were also investigated. No burst phase was observed with either substrate, indicating that product release does not limit the rate of catalysis in either direction. For the conversion of glutamate to methylaspartate, a single chemical step appeared to dominate the overall rate, whereas in the reverse direction a lag phase was observed, suggesting the accumulation of an intermediate, tentatively ascribed to glycyl radical and acrylate. The rates of formation and decay of this intermediate were also sufficiently rapid for it to be kinetically competent. When combined with information from previous mechanistic studies, these results allow a qualitative free energy profile to constructed for the reaction catalyzed by glutamate mutase.     

Dynamics of proton transfer and enzymatic activity

The rate-determining elementary reaction step, i.e. proton transfer from the chymotrypsin active centre to the scissile substrate bond has been studied in the present work. On the basis of our theoretical results a hypothesis was formulated to explain chymotrypsin enzymatic efficiency. After ES complex formation excited vibrational states are populated in the enzyme molecule. In the rate-determining elementary reaction step, the proton transfer takes place from the first excited vibrational state of the N-H bond in the imidazole group of His57. This proton transfer is realised by quantum mechanical tunneling mechanism.     

Analysis of the kinetic mechanism of halophilic NADP-dependent glutamate dehydrogenase

The amination of 2-oxoglutarate catalyzed by NADP-specific glutamate dehydrogenase (EC 1.4.1.4, L-glutamate:NADP+ oxidoreductase (deaminating)) from Halobacterium halobium has been analyzed by initial rate, graphical analysis, and product and competitive inhibition studies. Initial rate and graphical analysis reveal that a B term (representing 2-oxoglutarate) is not statistically necessary for an initial rate equation. However, the absence of a B term does not distinguish between ordered and random binding of NADPH and ammonia. The patterns of product inhibition by NADP+ and L-glutamate, and competitive inhibition by hydroxylamine and succinate permit deduction of the kinetic mechanism as ordered, with NADPH, 2-oxoglutarate and ammonia added in that order, and L-glutamate release preceding NADP+ release.     

A proposed proton shuttle mechanism for saccharopine dehydrogenase from Saccharomyces cerevisiae

Saccharopine dehydrogenase [N6-(glutaryl-2)-L-lysine:NAD oxidoreductase (L-lysine forming)] catalyzes the final step in the alpha-aminoadipate pathway for lysine biosynthesis. It catalyzes the reversible pyridine nucleotide-dependent oxidative deamination of saccharopine to generate alpha-Kg and lysine using NAD+ as an oxidizing agent. The proton shuttle chemical mechanism is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. In the direction of lysine formation, once NAD+ and saccharopine bind, a group with a pKa of 6.2 accepts a proton from the secondary amine of saccharopine as it is oxidized. This protonated general base then does not participate in the reaction again until lysine is formed at the completion of the reaction. A general base with a pKa of 7.2 accepts a proton from H2O as it attacks the Schiff base carbon of saccharopine to form the carbinolamine intermediate. The same residue then serves as a general acid and donates a proton to the carbinolamine nitrogen to give the protonated carbinolamine. Collapse of the carbinolamine is then facilitated by the same group accepting a proton from the carbinolamine hydroxyl to generate alpha-Kg and lysine. The amine nitrogen is then protonated by the group that originally accepted a proton from the secondary amine of saccharopine, and products are released. In the reverse reaction direction, finite primary deuterium kinetic isotope effects were observed for all parameters with the exception of V2/K(NADH), consistent with a steady-state random mechanism and indicative of a contribution from hydride transfer to rate limitation. The pH dependence, as determined from the primary isotope effect on DV2 and D(V2/K(Lys)), suggests that a step other than hydride transfer becomes rate-limiting as the pH is increased. This step is likely protonation/deprotonation of the carbinolamine nitrogen formed as an intermediate in imine hydrolysis. The observed solvent isotope effect indicates that proton transfer also contributes to rate limitation. A concerted proton and hydride transfer is suggested by multiple substrate/solvent isotope effects, as well as a proton transfer in another step, likely hydrolysis of the carbinolamine. In agreement, dome-shaped proton inventories are observed for V2 and V2/K(Lys), suggesting that proton transfer exists in at least two sequential transition states.     

Evidence for coupled motion and hydrogen tunneling of the reaction catalyzed by glutamate mutase

Glutamate mutase is one of a group of adenosylcobalamin-dependent enzymes that catalyze unusual isomerizations that proceed through organic radical intermediates generated by homolytic fission of the coenzyme's unique cobalt-carbon bond. These enzymes are part of a larger family of enzymes that catalyze radical chemistry in which a key step is the abstraction of a hydrogen atom from an otherwise inert substrate. To gain insight into the mechanism of hydrogen transfer, we previously used pre-steady-state, rapid-quench techniques to measure the alpha-secondary tritium kinetic and equilibrium isotope effects associated with the formation of 5'-deoxyadenosine when glutamate mutase was reacted with [5'-(3)H]adenosylcobalamin and L-glutamate. We showed that both the kinetic and equilibrium isotope effects are large and inverse, 0.76 and 0.72, respectively. We have now repeated these measurements using glutamate deuterated in the position of hydrogen abstraction. The effect of introducing a primary deuterium kinetic isotope effect on the hydrogen transfer step is to reduce the magnitude of the secondary kinetic isotope effect to a value close to unity, 1.05 +/- 0.08, whereas the equilibrium isotope effect is unchanged. The significant reduction in the secondary kinetic isotope effect is consistent with motions of the 5'-hydrogen atoms being coupled in the transition state to the motion of the hydrogen undergoing transfer, in a reaction that involves a large degree of quantum tunneling.