Pyridoxamine-pyruvate transaminase

For two transaminases the remaining unknown stereochemical parameter was determined by demonstrating an internal transfer of tritium (dialkyl amino acid transaminase) [28] or deuterium (pyridoxamine-pyruvate transaminase) [27] from the a-position of the substrate L-alanine to C-4 of the cofactor. Internal hydrogen transfer from the a-position of the substrate amino acid to C-4 of PLP has also been demonstrated for two of the abortive transamination reactions, those catalyzed by tryptophan synthase fi2 protein [32] and by aspartate-/8-decarboxylase [31]. In addition, the same phenomenon must occur in alanine transaminase, as deduced from the observation that the enzyme catalyzes exchange of the /8-hydrogens of... 

In pathway A (Figure 1), observed in Pseudomonas MA-1, pyridoxal (2) is produced either from pyridoxamine (15) by a transamination reaction with pyruvate catalyzed by pyridoxamine pyruvate transaminase,or from pyridoxine (1) by an oxidation reaction catalyzed by the FAD-dependent... 

Subsequent to this work, apoaspartate transaminase was used to assay the stereospecificity of a variety of other transaminases, all of which were shown to involve protonation/deprotonation at the C-4 Si face of the cofactor. These enzymes included pyridoxamine-pyruvate transaminase (EC 2.6.1.30) (26) and a-dialkylamino acid transaminase (27). L-Glutamate decarboxylase (EC 4.1.1.15) catalyzes an abortive transamination reaction when oc-methylglutamate is used as substrate, and this too was shown to occur with protonation at the Si face of C-4 in the intermediate 4d (28) as was the abnormal transamination of D-alanine by serine hydroxymethyltransferase (29). 

Pyridoxamine pyruvate transaminase, 21 Pyrimidine deaminases, 22 Pyrimidine nucleotides, 90 Pyruvate carboxylase, labeling of, 139 Pyruvate decarboxylase, 50, 51 Pyruvate kinase, 21, 241, 248, 299, 306, 307, 529, 548... 

Hydrolyze Schiff base Release a-keto acid and pyridoxamine phosphate Transfer of amino group from pyridoxamine phosphate to pyruvate, forming alanine occurs by reversal of these steps. Other transaminases use other a-ami no acids and a-keto acids. 

Amino acids NAD(P)H Pyridoxamine 5 -phosphate Pyruvate Amines Pyridoxal 5-phosphate dependent enzymes Dehydrogenases Transaminases Pyridoxal 5-phosphate dependent enzymes Amino acid decarboxylases... 

The same conclusion applies for L-alanine/pyruvate (2S7), pyridoxamine/pyru-vate (238), and dialkylamino acid transaminase (239). Second, small but significant amounts of direct hydrogen transfer (as either or H) between the a-carbon and C-4 have been detected during catalysis by the latter two enzymes (238, 239). Thus, the clear implication of these results is that a single active site base operating at the si face of the aldimine facilitates proton transfer between the a-carbon and C-4 [Eq. (47)]. 

Groups with special conformation were introduced into CD. This could get analogue enzyme model with stereoscopic selectivity. For example, pyridoxamine (PM) was bonded to the first hydroxyl group of p CD. The obtained modified CD could catalyze amino transfer reaction of a-ketone add. The reaction speed was 100 fold that of PM as catalyst and the stereoselectivity was shown during the catalysis [16]. If the PM group was linked to the main surface of -CD through double bonds, the transaminase model would be obtained. It was indicated from studies that when PM group was located on one side of CD, the substrate of meta substituted phenyl pyruvic add would be Unked as priority selection when it was located above the cavity of CD, the substrate of p-substituted phenyl pyruvic acid would be linked as priority selection [17]. 

Transaminases also belong to the group of transferases and catalyze desamination and amination reactions. Most amino acids are in equilibrium with the corresponding 0x0-carboxylic acid like alanine and pyruvate. The coenzyme of enzymatic transamination is pyridoxal-5 -phosphate 93, it is one of the most important coenzymes and is in effect the biologically active form of vitamin B6, which is converted to pyridoxamine 94 in this reaction. Besides transamination, pyridoxal-5 -phosphate 93 is also involved in decarboxylation and racemization processes. The aldehyde function of pyridoxal is reacting with the amine of an amino acid to form a Schiff base and thus invers-ing the acidity of the a-CH. The reversible transfer of an... 

In the described examples, the pyridoxamine was covalently attached to the polymer while in most real transaminase enzymes the pyridoxamine coenzyme forms a noncovalent active holoenzyme with the protein (apoenzyme). A new artificial transaminase mimic was developed, in which the pyridoxamine binds noncovalently and reversibly to the polymer. The pyridoxamine attached, for example, to a steroid side chain 99 or 100, together with modified PEI 101 (molecular weight of 60000 and 8.7% dodecyl chains) forms the artificial holoenzyme (Figure 38a). The transamination of pyruvic acid was accelerated 28000-fold with 99 + 101 compared to 10 000 with the covalent pyridoxamine-polymer 98 enzyme mimic. This was due to the fact that the noncovalent system 99 - -101 is more dynamic and therefore can adopt a more suitable geometry for the reaction. The artificial transaminase shows effective rate enhancements in converting the ketoacid into the amino acid, but also the pyridoxamine is converted to pyridoxal. The conversion to pyridoxamine is a necessary step in the catalytic cycle to achieve high turnovers however, this was still not possible with the noncovalent model system. It was observed that the reverse process is very slow and actually in all artificial models so far thermodynamically unfavorable. However, it was possible to use sacrificial amino acids at elevated temperatures (60 °C) that were decarboxy-lated to recycle the pyridoxal 102 to pyridoxamine 100 with modest turnover numbers of 81 (Figure 38b). " ... 

It has since been shown that reversible, non-enzymic transamina- tion occurs between pyridoxal and alanine and pyridoxamine and pyruvate in neutral, aqueous solutions, at 25° and in the absence of metal ions. (No evidence has yet been obtained for the presence rfj metal ions in purified mammalian transaminases.) Under thesi f conditions, no decarboxylation or racemisation of the amino add could be detected. From the results of kinetic and spectrophotometricl studies in this simple model system for transamination it has beenf shown that imines are formed rapidly and reversibly between the twtf pairs of reactants and that tautomerisation of the two SchifF s bascf intermediates is rate limiting in the overall transamination reactioni The reaction scheme is given in Figure 7. 

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