The K258R mutant of aspartate aminotransferase stabilizes the quinonoid intermediate.

Lys-258 of aspartate aminotransferase forms a Schiff base with pyridoxal phosphate and is responsible for catalysis of the 1,3-prototropic shift central to the transamination reaction sequence. Substitution of arginine for Lys-258 stabilizes the otherwise elusive quinonoid intermediate, as assessed by the long wavelength absorption bands observed in the reactions of this mutant with several amino acid substrates. The external aldimine intermediate is not detectable during reactions of this mutant with amino acids, although the inhibitor alpha-methylaspartate does slowly and stably form this species. These results suggest that external aldimine formation is one of the rate-determining steps of the reaction. The pyridoxamine-5'-phosphate-like enzyme form (330-nm absorption maximum) is unreactive toward keto acid substrates, and the coenzyme bound to this species is not dissociable from the protein.     

Citrobacter freundii tyrosine phenol-lyase: the role of asparagine 185 in modulating enzyme function through stabilization of a quinonoid intermediate

Asn185 is an invariant residue in all known sequences of TPL and of closely related tryptophanase and it may be aligned with the Asn194 in aspartate aminotransferase. According to X-ray data, in the holoenzyme and in the Michaelis complex Asn185 does not interact with the cofactor pyridoxal 5'-phosphate, but in the external aldimine a conformational change occurs which is accompanied by formation of a hydrogen bond between Asn185 and the oxygen atom in position 3 of the cofactor. The substitution of Asn185 in TPL by alanine results in a mutant N185A TPL of moderate residual activity (2%) with respect to adequate substrates, L-tyrosine and 3-fluoro-L-tyrosine. The affinities of the mutant enzyme for various amino acid substrates and inhibitors, studied by both steady-state and rapid kinetic techniques, were lower than for the wild-type TPL. This effect mainly results from destabilization of the quinonoid intermediate, and it is therefore concluded that the hydrogen bond between Asn185 and the oxygen at the C-3 position of the cofactor is maintained in the quinonoid intermediate. The relative destabilization of the quinonoid intermediate and external aldimine leads to the formation of large amounts of gem-diamine in reactions of N185A TPL with 3-fluoro-L-tyrosine and L-phenylalanine. For the reaction with 3-fluoro-L-tyrosine it was first possible to determine kinetic parameters of gem-diamine formation by the stopped-flow method. For the reactions of N185A TPL with substrates bearing good leaving groups the observed values of k(cat) could be accounted for by taking into consideration two effects: the decrease in the quinonoid content under steady-state conditions and the increase in the quinonoid reactivity in a beta-elimination reaction. Both effects are due to destabilization of the quinonoid and they counterbalance each other. Multiple kinetic isotope effect studies on the reactions of N185A TPL with suitable substrates, L-tyrosine and 3-fluoro-L-tyrosine, show that the principal mechanism of catalysis, suggested previously for the wild-type enzyme, does not change. In the framework of this mechanism the observed considerable decrease in k(cat) values for reactions of N185A TPL with L-tyrosine and 3-fluoro-L-tyrosine may be ascribed to participation of Asn185 in additional stabilization of the keto quinonoid intermediate.     

Aminotransferase activity and bioinformatic analysis of 1-aminocyclopropane-1-carboxylate synthase

The mechanistic fate of pyridoxal phosphate (PLP)-dependent enzymes diverges after the quinonoid intermediate. 1-Aminocyclopropane-1-carboxylate (ACC) synthase, a member of the alpha family of PLP-dependent enzymes, is optimized to direct electrons from the quinonoid intermediate to the gamma-carbon of its substrate, S-adenosyl-L-methionine (SAM), to yield ACC and 5'-methylthioadenosine. The data presented show that this quinonoid may also accept a proton at C(4)' of the cofactor to yield alpha-keto acids and the pyridoxamine phosphate (PMP) form of the enzyme when other amino acids are presented as alternative substrates. Addition of excess pyruvate converts the PMP form of the enzyme back to the PLP form. C(alpha)-deprotonation from L-Ala is shown by NMR-monitored solvent exchange to be reversible with a rate that is less than 25-fold slower than that of deprotonation of SAM. The rate-determining step for transamination follows the formation of the quinonoid intermediate. The rate-determining step for alpha, gamma-elimination from enzyme-bound SAM is likewise shown to occur after C(alpha)-deprotonation, and the quinonoid intermediate accumulates during this reaction. BLAST searches, sequence alignments, and structural comparisons indicate that ACC synthases are evolutionarily related to the aminotransferases. In agreement with previously published reports, an absence of homology was found between the alpha and beta families of the PLP-dependent enzyme superfamily.     

Novel biosynthetic routes to non-proteinogenic amino acids as chiral pharmaceutical intermediates

Transaminases catalyse the reversible transfer of amino and keto groups between an amino acid and keto acid substrate pair. Many bacterial transaminases accept a wide array of keto acids as amino acceptors and are useful as commercial biocatalysts in the preparation of amino acids. Since the reaction equilibrium typically lies close to unity, several approaches have been described to improve upon the 50% product yield, using additional enzymes. The present work describes an efficient means to significantly increase product yield in transamination using the aromatic transaminase of Escherichia coli encoded by the tyrB gene, with -aspartate as the amino donor. This is achieved by the introduction of the alsS gene encoding the acetolactate synthase of Bacillus subtilis, which eliminates pyruvate and alanine produced as a by-product of aspartate transamination. The biosynthesis of the non-proteinogenic amino acid -2-aminobutyrate is described using a recombinant strain of E. coli containing the cloned tyrB and alsS genes. The strain additionally carries the cloned ilvA gene of E. coli encoding threonine deaminase to produce the substrate 2-ketobutyrate from -threonine. An alternate coupled process uses lysine -aminotransferase in concert with a transaminase using -glutamate as the amino donor.     

Stereospecificity of reactions catalyzed by bacterial D-amino acid transaminase

The spectral shift from 420 to 338 nm when pure bacterial D-amino acid transaminase binds D-amino acid substrates is also exhibited in part by high concentrations of L-amino acids (L-alanine and L-glutamate) but not by simple dicarboxylic acids or monoamines. Slow processing of L-alanine to D-alanine was observed both by coupled enzymatic assays using D-amino acid oxidase and by high pressure liquid chromatography analysis employing an optically active chromophore (Marfey's reagent). When the acceptor for L-alanine was alpha-ketoglutarate, D-glutamate was also formed. This minor activity of the transaminase involved both homologous (L-alanine and D-alanine) and heterologous (L-alanine and D-glutamate) substrate pairs and was a function of the nature of the keto acid acceptor. In the presence of alpha-ketoisovalerate, DL-alanine was almost completely processed to D-valine; within the limits of the assay no L-valine was detected. With alpha-ketoisocaproate, 90% of the DL-alanine was converted to D-leucine. In the mechanism of this transaminase reaction, there may be more stereoselective constraints for the protonation of the quinonoid intermediate during the second half-reaction of the transamination reaction, i.e. the donation of the amino group from the pyridoxamine 5'-phosphate coenzyme to a second keto acid acceptor, than during removal of the alpha proton in the initial steps of the reaction pathway. Thus, with this D-amino acid transaminase, the discrete steps of transamination ensure fidelity of the stereospecificity of reaction pathway.     

AROMATIC AMINOTRANSFERASE ACTIVITY OF RAT BRAIN CYTOPLASMIC AND MITOCHONDRIAL ASPARTATE AMINOTRANSFERASES

Abstract— Mitochondrial and cytoplasmic forms of aspartate aminotransferase were purified from rat brain homogenates and tested for their ability to catalyze transamination of various aromatic amino acids. The mitochondrial enzyme exhibited activity toward tyrosine and phenylalanine with 2-oxoglutar-ate as acceptor, although the specific activities were less than 1% of the corresponding aspartate activity when all substrates were 10 mM. Even less activity was seen with DOPA, 5-hydroxytryptophan and tryptophan. The cytoplasmic aspartate aminotransferase was active toward tryptophan, 5-hydroxytryptophan and DOPA, but these transaminations were favored by pyruvate or oxaloacetate rather than 2-oxoglutarate as keto acid. Based on co-migration of aromatic activities with the respective aspartate aminotransferases during isoelectric focusing and based on equal sensitivities of aromatic transamination and aspartate transamination to inhibition by vinylglycine, it was concluded that all activities resided in the aspartate aminotransferase enzymes. Some doubt exists, however, as to the physiological significance of these alternate activities in view of the requirement that aromatic amino acids must compete with aspartate for transamination by these enzymes.     

The mechanism of action of ethanolamine ammonia-lyase, an adenosylcobalamin-dependent enzyme. Evidence that the hydrogen transfer mechanism involves a second intermediate hydrogen carrier in addition to the cofactor

Ethanolamine ammonia-lyase catalyzes the adenosylcobalamin (AdoCbl)-dependent conversion of ethanolamine to acetaldehyde and ammonia. During this reaction, a hydrogen atom migrates from the carbinol carbon of ethanolamine to the methyl carbon of acetaldehyde. Previous studies have shown that this migrating hydrogen equilibrates with the hydrogens on the 5'-(cobalt-linked) carbon of the cofactor. On the basis of those studies, a two-step mechanism for hydrogen transfer has been postulated in which the migrating hydrogen is first transferred from the substrate to the cofactor, then in a subsequent step is returned from the cofactor to the product. We now show that this migrating hydrogen is transferred not only to the cofactor, but also to a second acceptor at the active site. Hydrogens on this acceptor do not exchange with water during the course of the reaction, but are released to water when the enzyme is denatured. The catalytic significance of this second hydrogen acceptor was demonstrated by the findings that the transfer of hydrogen to this acceptor required both AdoCbl and active enzyme and that hydrogen at the second acceptor site could be washed out by unlabeled ethanolamine. On the basis of these results, we propose an expanded hydrogen transfer mechanism in which AdoCbl and the second acceptor site serve as alternative intermediate hydrogen carriers during the course of ethanolamine deamination.     

Rerouting Citrate Metabolism in Lactococcus lactis to Citrate-Driven Transamination

Oxaloacetate is an intermediate of the citrate fermentation pathway that accumulates in the cytoplasm of Lactococcus lactis ILCitM(pFL3) at a high concentration due to the inactivation of oxaloacetate decarboxylase. An excess of toxic oxaloacetate is excreted into the medium in exchange for citrate by the citrate transporter CitP (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:4049-4056, 2011). In this study, transamination of amino acids with oxaloacetate as the keto donor is described as an additional mechanism to relieve toxic stress. Redirection of the citrate metabolic pathway into the transamination route in the presence of the branched-chain amino acids Ile, Leu, and Val; the aromatic amino acids Phe, Trp, and Tyr; and Met resulted in the formation of aspartate and the corresponding α-keto acids. Cells grown in the presence of citrate showed 3.5 to 7 times higher transaminase activity in the cytoplasm than cells grown in the absence of citrate. The study demonstrates that transaminases of L. lactis accept oxaloacetate as a keto donor. A significant fraction of 2-keto-4-methylthiobutyrate formed from methionine by citrate-driven transamination in vivo was further metabolized, yielding the cheese aroma compounds 2-hydroxy-4-methylthiobutyrate and methyl-3-methylthiopropionate. Reducing equivalents required for the former compound were produced in the citrate fermentation pathway as NADH. Similarly, phenylpyruvate, the transamination product of phenylalanine, was reduced to phenyllactate, while the dehydrogenase activity was not observed for the branched-chain keto acids. Both α-keto acids and α-hydroxy acids are known substrates of CitP and may be excreted from the cell in exchange for citrate or oxaloacetate.     

Uronate isomerase: a nonhydrolytic member of the amidohydrolase superfamily with an ambivalent requirement for a divalent metal ion

Uronate isomerase, a member of the amidohydrolase superfamily, catalyzes the isomerization of D-glucuronate and D-fructuronate. During the interconversion of substrate and product the hydrogen at C2 of D-glucuronate is transferred to the pro-R position at C1 of the product, D-fructuronate. The exchange of the transferred hydrogen with solvent deuterium occurs at a rate that is 4 orders of magnitude slower than the interconversion of substrate and product. The enzyme catalyzes the elimination of fluoride from 3-deoxy-3-fluoro-D-glucuronate. These results have been interpreted to suggest a chemical reaction mechanism in which an active site base abstracts the proton from C2 of D-glucuronate to form a cis-enediol intermediate. The conjugate acid then transfers this proton to C1 of the cis-enediol intermediate to form D-fructuronate. The loss of fluoride from 3-deoxy-3-fluoro-D-glucuronate is consistent with a stabilized carbanion at C2 of the substrate during substrate turnover. The slow exchange of the transferred hydrogen with solvent water is consistent with a shielded conjugate acid after abstraction of the proton from either D-glucuronate or D-fructuronate during the isomerization reaction. This conclusion is supported by the competitive inhibition of the enzymatic reaction by D-arabinaric acid and the monohydroxamate derivative with Ki values of 13 and 670 nM, respectively. There is no evidence to support a hydride transfer mechanism for uronate isomerase. The wild type enzyme was found to contain 1 equiv of zinc per subunit. The divalent cation could be removed by dialysis against the metal chelator, dipicolinate. However, the apoenzyme has the same catalytic activity as the Zn-substituted enzyme and thus the divalent metal ion is not required for enzymatic activity. This is the only documented example of a member in the amidohydrolase superfamily that does not require one or two divalent cations for enzymatic activity.     

Reactions of 3'-O-methylpyridoxal 5'-phosphate in aspartate aminotransferase

The reaction of 3'-O-methylpyridoxal 5'-phosphate bound into the active site of aspartate aminotransferase with the substrate L-aspartate has been investigated. This methylated coenzyme is a very poor catalyst but it does function slowly to produce normal products of a transamination half-reaction. At pH 8.5 and above the characteristic absorption band of a quinonoid intermediate appears rapidly and becomes very intense when the aspartate concentration is raised to 2 M. At pH 6 the quinonoid band is not seen, but the conversion of the methylated coenzyme into 3'-O-methylpyridoxamine 5'-phosphate is about 7 times faster than at high pH with the pH dependence being determined by an apparent pKa of 8.1 at 30 degrees C. We suggest that the active site containing the methylated coenzyme carries a net charge 1 unit more positive than that of native enzyme. This causes a loss of some other proton from the active site and could leave the catalytic lysine-258 deprotonated in the quinonoid species. This may explain its inability to react rapidly. We have measured the spectral band shapes of the quinonoid species studied here and have compared it with that seen with native enzyme. Because of the close similarity we conclude that during normal transamination the proton bound to the imine nitrogen probably shifts onto the phenolic oxygen prior to or synchronously with the formation of the observed quinonoid species.     

Serine hydroxymethyltransferase: mechanism of the racemization and transamination of D- and L-alanine

The reaction specificity and stereochemical control of Escherichia coli serine hydroxymethyltransferase were investigated with D- and L-alanine as substrates. An active-site H228N mutant enzyme binds both D- and L-alanine with Kd values of 5 mM as compared to 30 and 10 mM, respectively, for the wild-type enzyme. Both wild-type and H228N enzymes form quinonoid complexes absorbing at 505 nm by catalyzing the loss of the alpha-proton from both D- and L-alanine. Racemization and transamination reactions were observed to occur with both alanine isomers as substrates. The relative rates of these reactions are quinonoid formation greater than alpha-proton solvent exchange greater than racemization greater than transamination. The observation that the rate of quinonoid formation with either alanine isomer is an order of magnitude faster than solvent exchange suggests that the alpha-protons from both D- and L-alanine are transferred to base(s) on the enzyme. The rate of racemization is 2 orders of magnitude slower than the formation of the quinonoid complexes. This latter difference in rate suggests that the quinonoid complexes formed from D- and L-alanine are not identical. The difference in structure of the two quinonoid complexes is proposed to be the active-site location of the alpha-protons lost from the two alanine isomers, rather than two orientations of the pyridoxal phosphate ring. The results are consistent with a two-base mechanism for racemization.     

Biosynthetic origin of hydrogen atoms in the lipase inhibitor lipstatin

The lipase inhibitor lipstatin is biosynthesized in Streptomyces toxytricini via condensation of a C(14) precursor and a C(8) precursor, which are both obtained from fatty acid catabolism. To study the mechanism of this reaction in more detail, S. toxytricini was grown in medium containing a mixture of U-(13)C,U-(2)H-lipids and unlabeled sunflower oil or in a medium containing 70% D(2)O. Lipstatin was isolated and analyzed by (1)H,(2)H, and (13)C NMR spectroscopy. Hydrogen atoms at C-2, C-3, and C-4 of lipstatin were found to be derived from solvent protons. The formation of the lipstatin precursor 3-hydroxy-Delta(5,8)-tetradecadienoyl-CoA by beta oxidation of linoleic acid explains the incorporation of solvent hydrogen into the 4 position of lipstatin. The hydrogen in position 3 of lipstatin is most probably introduced from solvent by proton/deuterium exchange of a redox cofactor involved in the reduction of the keto group in the branched chain beta keto acid arising by a decarboxylative condensation. The incorporation of solvent hydrogen at position 2 can be explained by epimerization of a chiral intermediate at C-2 and C-3. Epimerization may involve a dehydration-rehydration mechanism.     

Stereospecific labilization of the C-4' pro-S hydrogen of pyridoxamine 5'-phosphate in aspartate aminotransferase

In the course of a half-reaction of enzymic transamination, the aldimine adduct formed between the coenzyme pyridoxal 5'-phosphate and the amino acid substrate tautomerizes to the ketimine intermediate which is then hydrolyzed to the oxo acid product and the pyridoxamine 5'-phosphate form of the enzyme. In the reverse half-reaction the tautomerization is initiated by the removal of a proton from the pro-S position at C-4' of the PMP moiety of the ketimine intermediate. The present study investigates the question whether the pro-S hydrogen at C-4' of PMP is labilized by its active site environment independently of the formation of the ketimine intermediate, i.e. in the absence of substrate. Reconstitution of apoaspartate aminotransferase (mitochondrial isoenzyme from chicken) with [4'-3H] PMP results indeed in a stereospecific exchange of pro-S 3H with solvent water. The exchange follows first order kinetics (t 1/2 = 23 min at pH 7.5 and 25 degrees C). Unbound PMP showed no measurable exchange. Rigorous control experiments excluded the possibility that the observed exchange was due to a transamination reaction of the enzyme with contaminating oxo acid substrates. The newly observed stereospecific exchange reaction allows to investigate the acid/base properties of C-4' and the modulating effects of its active site environment independently of the preceding and following steps of enzymic transamination.     

Intermolecular hydrogen transfer catalyzed by a flavodehydrogenase, bakers' yeast flavocytochrome b2

Bakers' yeast flavocytochrome b2 is a flavin-dependent L-2-hydroxy acid dehydrogenase which also exhibits transhydrogenase activity. When a reaction takes place between [2-3H]lactate and a halogenopyruvate, tritium is found in water and at the halogenolactate C2 position. When the halogenopyruvate undergoes halide ion elimination, tritium is also found at the C3 position of the resulting pyruvate. The amount tau of this intermolecular tritium transfer depends on the initial keto acid-acceptor concentration. At infinite acceptor concentration, extrapolation yields a maximal transfer of 97 +/- 11%. This indicates that the hydroxy acid-derived hydrogen resides transiently on enzyme monoprotic heteroatoms and that exchange with bulk solvent occurs only at the level of free reduced enzyme. Using a minimal kinetic scheme, the rate constant for hydrogen exchange between Ered and solvent is calculated to be on the order of 10(2) M-1 S-1, which leads to an estimated pK approximately equal to 15 for the ionization of the substrate-derived proton while on the enzyme. It is suggested that this hydrogen could be shared between the active site base and Flred N5 anion. It is furthermore shown that some tritium is incorporated into the products when the transhydrogenation is carried out in tritiated water. Finally, with [2-2H]lactate-reduced enzyme, a deuterium isotope effect is observed on the rate of bromopyruvate disappearance. Extrapolation to infinite bromopyruvate concentration yields DV = 4.4. An apparent inverse isotope effect is determined for bromide ion elimination. These results strengthen the idea that oxidoreduction and elimination pathways involve a common carbanionic intermediate.     

Aspartate aminotransferase isotope exchange reactions: implications for glutamate/glutamine shuttle hypothesis

Aspartate aminotransferase (AAT) catalyzes amino group transfer from glutamate (Glu) or aspartate (Asp) to a keto acid acceptor-oxaloacetate (OA) or alpha-ketoglutarate (KG), respectively. Data presented here show that AAT catalyzes two partial reactions resulting in isotope exchange between 3H-labeled Glu or 3H-labeled Asp and the cognate keto acid in the absence of the keto acid acceptor required for the net reaction. Tritiated keto acid product was detected by release of 3H2O from C-3 during base-induced enolization. Tritium released directly from C-2 (or C-3) by the enzyme was also evaluated and is a small fraction of that released because of exchange to the keto acid pool. Exchange is dependent on AAT concentration, time-dependent, proportional to the amino-to-keto acid ratio, and blocked by aminooxyacetate (AOA), an AAT inhibitor. Enzymatic conversion of [3H]KG to Glu by glutamic dehydrogenase (GDH) or of [3H]OA to malate by malic dehydrogenase (MDH) "protects" the label from release by base, showing that base-induced isotope release is from keto acid rather than a result of release during the exchange process. AAT isotope exchange is discussed in the context of the glutamate/glutamine shuttle hypothesis for astrocyte/neuron carbon cycling.     

Lysine-313 of 5-aminolevulinate synthase acts as a general base during formation of the quinonoid reaction intermediates

5-Aminolevulinate synthase catalyzes the condensation of glycine and succinyl-CoA to form CoA, carbon dioxide, and 5-aminolevulinate. This represents the first committed step of heme biosynthesis in animals and some bacteria. Lysine 313 (K313) of mature murine erythroid 5-aminolevulinate synthase forms a Schiff base linkage to the pyridoxal 5'-phosphate cofactor. In the presence of glycine and succinyl-CoA, a quinonoid intermediate absorption is transiently observed in the visible spectrum of purified murine erythroid ALAS. Mutant enzymes with K313 replaced by glycine, histidine, or arginine exhibit no spectral evidence of quinonoid intermediate formation in the presence of glycine and succinyl-CoA. The wild-type 5-aminolevulinate synthase additionally forms a stable quinonoid intermediate in the presence of the product, 5-aminolevulinate. Only conservative mutation of K313 to histidine or arginine produces a variant that forms a quinonoid intermediate with 5-aminolevulinate. The quinonoid intermediate absorption of these mutants is markedly less than that of the wild-type enzyme, however. Whereas the wild-type enzyme catalyzes loss of tritium from [2-3H2]-glycine, mutation of K313 to glycine results in loss of this activity. Titration of the quinonoid intermediate formed upon binding of 5-aminolevulinate to the wild-type enzyme indicated that the quinonoid intermediate forms by transfer of a single proton with a pK of 8.1 +/- 0.1. Conservative mutation of K313 to histidine raises this value to 8.6 +/- 0.1. We propose that K313 acts as a general base catalyst to effect quinonoid intermediate formation during the 5-aminolevulinate synthase catalytic cycle.     

Insights into the catalytic mechanism of tyrosine phenol-lyase from X-ray structures of quinonoid intermediates

Amino acid transformations catalyzed by a number of pyridoxal 5'-phosphate (PLP)-dependent enzymes involve abstraction of the Calpha proton from an external aldimine formed between a substrate and the cofactor leading to the formation of a quinonoid intermediate. Despite the key role played by the quinonoid intermediates in the catalysis by PLP-dependent enzymes, limited accurate information is available about their structures. We trapped the quinonoid intermediates of Citrobacter freundii tyrosine phenol-lyase with L-alanine and L-methionine in the crystalline state and determined their structures at 1.9- and 1.95-A resolution, respectively, by cryo-crystallography. The data reveal a network of protein-PLP-substrate interactions that stabilize the planar geometry of the quinonoid intermediate. In both structures the protein subunits are found in two conformations, open and closed, uncovering the mechanism by which binding of the substrate and restructuring of the active site during its closure protect the quinonoid intermediate from the solvent and bring catalytically important residues into positions suitable for the abstraction of phenol during the beta-elimination of L-tyrosine. In addition, the structural data indicate a mechanism for alanine racemization involving two bases, Lys-257 and a water molecule. These two bases are connected by a hydrogen bonding system allowing internal transfer of the Calpha proton.     

Kinetic studies with the use of proton-magnetic-resonance spectroscopy of the specific alpha-deuteration of amino acids by Escherichia coli aspartate aminotransferase.

Escherichia coli aspartate aminotransferase was exposed to aspartate or phenylalanine without oxo acid in buffered 2H2O. The alpha-hydrogen of the amino acids underwent first-order exchange with respect to both substrate and enzyme. P.m.r. spectroscopy gave consistent reaction-rate constants. The deuterium-exchange rate was only moderately increased by addition of oxo acids and was of the same order as the transamination rate. No beta-deuteration was observed. The C(alpha)-H-bond-breaking step is discussed as a part of the entire transamination mechanism.     

Kinemage of action - proposed reaction mechanism of glutamate-1-semialdehyde aminomutase at an atomic level

Glutamate-1-semialdehyde aminomutase (GSAM), a key enzyme in tetrapyrrole cofactor biosynthesis, performs a unique transamination on a single substrate. The substrate, glutamate-1-semialdehyde (GSA), undergoes a reaction that exchanges the position of an amine and a carbonyl group to produce 5-aminolevulinic acid (ALA). This transamination reaction is unique in the fact that is does not require an external cofactor to act as a nitrogen donor or acceptor in this transamination reaction. One of the other remarkable features of the catalytic mechanism is the release free in the enzyme active site of the intermediate 4,5-diaminovaleric acid (DAVA). The action of a gating loop prevents the escape of DAVA from the active site. In a MD simulation approach, using snapshots provided by X-ray crystallography and protein crystal absorption spectrometry data, the individual catalytic steps in this unique intramolecular transamination have been elucidated.     

The mechanism of alpha-proton isotope exchange in amino acids catalysed by tyrosine phenol-lyase. What is the role of quinonoid intermediates?

To shed light on the mechanism of isotopic exchange of alpha-protons in amino acids catalyzed by pyridoxal phosphate (PLP)-dependent enzymes, we studied the kinetics of quinonoid intermediate formation for the reactions of tyrosine phenol-lyase with L-phenylalanine, L-methionine, and their alpha-deuterated analogues in D2O, and we compared the results with the rates of the isotopic exchange under the same conditions. We have found that, in the L-phenylalanine reaction, the internal return of the alpha-proton is operative, and allowing for its effect, the exchange rate is accounted for satisfactorily. Surprisingly, for the reaction with L-methionine, the enzymatic isotope exchange went much faster than might be predicted from the kinetic data for quinonoid intermediate formation. This result allows us to suggest the existence of an alternative, possibly concerted, mechanism of alpha-proton exchange.