Pyridoxal-5'-phosphate-dependent enzymes catalyze manifold reactions in the metabolism of amino acids. A comprehensive comparison of amino acid sequences has shown that most of these enzymes can be assigned to one of three different families of homologous proteins. The sequences of the enzymes of each family were aligned and their homology confirmed by profile analysis. Scrutiny of the reactions catalyzed by the enzymes showed that their affiliation with one of the three structurally defined families correlates in most cases with their regio-specificity.In the largest family, the covalency changes of the substrate occur at the same carbon atom that carries the amino group forming the irnine linkage with the coenzyme. This family was thus named a family. It comprises glycine hydroxymethyltransferase, glycine C-acetyltransferase, 5-aminulevulinate synthase, 8-amino-7-oxononanoate synthase, all aminotransferases (with the possible exception of subgroup 111), a number of other enzymes relatively closely related with the aminotransferases and very likely a certain group of amino acid decarboxylases as well as tryptophanase and tyrosine phenol-lyase which, however, catalyze p-elimination reactions. The p family includes L-and Dserine dehydratase, threonine dehydratase, the /? subunit of tryptophan synthase, threonine synthase and cysteine synthase. These enzymes catalyze p-replacement or p-elimination reactions. The y family incorporates 0-succinylhomoserine (thio1)-lyase, 0-acetylhomoserine (thio1)-lyase, and cystathionine y-lyase, which catalyze y-replacement or y-elimination reactions, as well as cystathionine P-lyase.The a and y family might be distantly related with one another, but are clearly not homologous with the / 3 family. Apparently, the primordial pyridoxal-5'-phosphate-dependent enzymes were regio-specific catalysts, which first specialized for reaction specificity and then for substrate specificity. The following pyridoxal-5'-phosphate-dependent enzymes seem to be unrelated with the a, /? or y family by the criterion of profile analysis: alanine racemase, selenocysteine synthase, and many amino acid decarboxylases. These enzymes may represent yet other families of B, enzymes.Pyridoxal-5'-phosphate-dependent enzymes (B, enzymes) catalyze such a wide variety of transformations of amino acids that they are found in no fewer than four out of the total six EC classes of enzymes (Enzyme Nomenclature, 1992). This paper is part of a study on the evolutionary relationships among these multifarious enzymes. Previously, we have found that most if not all aminotransferases constitute a group of homologous proteins . Profile analysis, an algorithm for the detection of distant relationships between amino acid sequences (Gribskov et al., 1990), showed, however, that the evolutionary relationships extend beyond the group of aminotransferases and include other B, enzymes Mehta and Christen, 1994). This study compares the currently known amino acid sequences of B, enzymes and shows that many of them belong to one of th...

Comparison of the amino acid sequences of nine different pyridoxal‐5′‐phosphate‐dependent amino acid decarboxylases indicated that they can be subdivided into four different groups that seem to be evolutionarily unrelated to each other. Group I is represented by glycine decarboxylase, a component of a multienzyme system; group II comprises glutamate, histidine, tyrosine, and aromatic‐l‐amino‐acid decarboxylases; group III, procaryotic ornithine and lysine decarboxylase as well as the procaryotic biodegradative type of arginine decarboxylase; group IV, eucaryotic ornithine and arginine decarboxylase as well as the procaryotic biosynthetic type of arginine decarboxylase and diaminopimelate decarboxylase. (N–1) profile analysis, a more stringent application of profile analysis, established the homology among the enzymes of each group. A search with the profile of group II indicated a distant relationship with aminotransferases and thus with the α family of pyridoxal‐5′‐phosphate‐dependent enzymes. No evidence was obtained that groups I, III and IV were related with other pyridoxal‐5′‐phosphate‐dependent enzymes or any other protein in the databse. Unlike the aminotransferases, which, with few possible exceptions, costitute a single group of homologous proteins, the amino acid decarboxylases, by the criterion of profile analysis, have evolved along multiple lineages, in some cases even if they have the same substrate specificity.

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The polypeptide binding and release cycle of the molecular chaperone DnaK (Hsp70) of Escherichia coli is regulated by the two co-chaperones DnaJ and GrpE. Here, we show that the DnaJ-triggered conversion of DnaK.ATP (T state) to DnaK.ADP.Pi (R state), as monitored by intrinsic protein fluorescence, is monophasic and occurs simultaneously with ATP hydrolysis. This is in contrast with the T-->R conversion in the absence of DnaJ which is biphasic, the first phase occurring simultaneously with the hydrolysis of ATP (Theyssen, H., Schuster, H.-P., Packschies, L., Bukau, B., and Reinstein, J. (1996) J. Mol. Biol. 263, 657-670). Apparently, DnaJ not only stimulates ATP hydrolysis but also couples it with conformational changes of DnaK. In the absence of GrpE, DnaJ forms a tight ternary complex with peptide.DnaK.ADP.Pi (Kd = 0.14 microM). However, by monitoring complex formation between DnaK (1 microM) and a fluorophore-labeled peptide in the presence of ATP (1 mM), DnaJ (1 microM), and varying concentrations of the ADP/ATP exchange factor GrpE (0.1-3 microM), substoichiometric concentrations of GrpE were found to shift the equilibrium from the slowly binding and releasing, high-affinity R state of DnaK completely to the fast binding and releasing, low-affinity T state and thus to prevent the formation of a long lived ternary DnaJ. substrate.DnaK.ADP.Pi complex. Under in vivo conditions with an estimated chaperone ratio of DnaK:DnaJ:GrpE = 10:1:3, both DnaJ and GrpE appear to control the chaperone cycle by transient interactions with DnaK.

and Arg 386 in substrate binding, the effects of their substitution on the activity toward long chain monocarboxylic (norleucine/2-oxocaproic acid) and aromatic substrates diverged. Whereas the R292K mutation did not impair the aminotransferase activity toward these substrates, the effect of the R386K substitution was similar to that on the activity toward dicarboxylic substrates. All three mutant enzymes catalyzed as side reactions the ␤-decarboxylation of L-aspartate and the racemization of amino acids at faster rates than the wild-type enzyme. The changes in reaction specificity were most pronounced in aspartate aminotransferase R292K, which decarboxylated L-aspartate to L-alanine 15 times faster (k cat ‫؍‬ 0.002 s ؊1 ) than the wild-type enzyme. The rates of racemization of L-aspartate, L-glutamate, and L-alanine were 3, 5, and 2 times, respectively, faster than with the wild-type enzyme. Thus, Arg 3 Lys substitutions in the active site of aspartate aminotransferase decrease aminotransferase activity but increase other pyridoxal 5-phosphate-dependent catalytic activities. Apparently, the reaction specificity of pyridoxal 5-phosphate-dependent enzymes is not only achieved by accelerating the specific reaction but also by preventing potential side reactions of the coenzyme substrate adduct.The pyridoxal 5Ј-phosphate (PLP) 1 -dependent enzymes that catalyze transformations of amino acids (for a recent review, see Ref. 1) constitute a few families of evolutionarily related enzymes (2). The member enzymes of such a family use the same protein scaffold to catalyze quite diverse reactions. Apparently, subtle structural differences underlie their catalytic specificity.Aspartate aminotransferase (AspAT) is probably the most extensively studied PLP-containing enzyme. It catalyzes the reversible transamination of the dicarboxylic L-amino acids, aspartate and glutamate, and the corresponding 2-oxo acids, oxalacetate and 2-oxoglutarate. During the catalytic cycle, the cofactor shuttles between the PLP and the pyridoxamine 5Ј-phosphate (PMP) forms. High resolution x-ray crystallographic analyses (3-6) in conjunction with site-directed mutagenesis studies (7-14) have elucidated the role of several active-site residues. The specificity for dicarboxylic amino acids appears to be based mainly on two active-site arginine residues (Fig.