Amino acid sequences pyridoxal phosphate

Like homogeneous catalysis, the removal of a-hydrogen of the amino acid fragment by OH ions, the local concentration of which is apparently high in the polymer phase, is probably the rate-determining step of heterogeneous racemization. Under similar conditions, the rate of a-amino acid racemization decreases in the sequence Ala = Ser>Phe>Nva>Lys>Val, and correlates with the rate of substrate racemization in the presence of Schiff bases and transamination of amino acids by pyridoxal phosphate. 

Katunuma and coworkers (1971) described a protease in the rat that hydrolyzes the apoenzymes of a number of pyridoxal phosphate-dependent enzymes it has no effect on other proteins or the holoenzymes. Presumably, it attacks the conserved amino acid sequence around the active lysine residue to which the internal Schiff base is formed. The activity ofthe enzyme is increased some 10- to 20-fold in vitamin Be deficiency, suggesting that its function is to degrade those enzymes that lose their coenzyme more readily, and so make more pyridoxal phosphate available for use by other enzymes. There is also evidence that some pyridoxal phosphate-dependent apoenzymes are modified to become incapable of activation by pyridoxal phosphate, although retaining immunological cross-reactivity with the normal form of the enzyme in vitamin Be deficiency (Nagata and Okada, 1985). 

Aspartate aminotransferase is the prototype of a large family of PLP-dependent enzymes. Comparisons of amino acid sequences as well as several three-dimensional structures reveal that almost all transaminases having roles in amino acid biosynthesis are related to aspartate aminotransferase by divergent evolution. An examination of the aligned amino acid sequences reveals that two residues are completely conserved. These residues are the lysine residue that forms the Schiff base with the pyridoxal phosphate cofactor (lysine 258 in aspartate aminotransferase) and an arginine residue that interacts with the a-carboxylate group of the ketoacid (see Figure 23.11). 

The presence of pyridoxal phosphate confers specific absorbance properties onto the holoenzyme. The absorption maximum is at 330 mp, a wavelength far removed from that of the Schiff base. Therefore, the combination of apoenzyme and coenzyme in the active phosphorylase molecule does not involve the formation of a Schiff base. Krebs and others studied the site of attachment of the phosphorus in the phosphorylase molecule by preparing P-labeled phosphorylase. After the enzyme was treated with trypsin, it became clear that the phosphorus was present in a basic hexa-peptide containing two basic amino acids. The hexa-peptide has the following amino acid sequence lysine, glutamine, isoleucine, serine phosphate, valine, and arginine. 

There is an important biochemical counterpart of the deamination reaction that utilizes pyridoxal phosphate, 7, as the aldehyde. Each step in the sequence is catalyzed by a specific enzyme. The a-amino group of the amino acid combines with 7 and is converted to a keto acid. The resulting pyridoxamine then reacts to form an imine with a different a-keto acid, resulting in formation of a new a-amino acid and regenerating 7. The overall process is shown in Equation 25-6 and is called transamination. It is a key part of the process whereby amino acids are metabolized. 

Fig. 8.1. Two-dimensional schematic representation of the structure of the adenine nucleotide carrier. The line represents the amino acid chain of the protein and all numbers on or within the line represent the number of the amino acids in the linear sequence. The black dots are cysteine residues, about which there is significant sequence homology [186]. The helical regions are segments of hydrophobic amino acids thought to span the membrane [186]. The CAT arrow represents the site of photoaffinity labelling of an azido derivative of atractyloside [189]. The open circles are lysine residues which react with pyridoxal phosphate in intact mitochondria or submitochondrial particles [190,191].

Our laboratory has studied the stereochemistry of methyl group formation in a number of a, 0 elimination reactions of amino acids catalyzed by pyridoxal phosphate enzymes. The reactions include the conversions of L-serine to pyruvate with tryptophan synthase 02 protein (78) and tryptophanase (79), of L-serine and l-tyrosine with tyrosine phenol-lyase (80), and l-cystine with S-alkylcysteine lyase (81). In the latter study, the stereospecific isotopically labeled L-cystines were obtained enzymatically by incubation of L-serines appropriately labeled in the 3-position with the enzyme O-acetyl serine sulfhy-drase (82). The serines tritiated in the 3-position were prepared enzymatically starting from [l-3H]glucose and [l-3H]mannose by a sequence of reactions of known stereochemistry (81). The cysteines were then incubated with 5-alkyl-cysteine lyase in 2H20 as outlined in Scheme 19. The pyruvate was trapped as lactate, which was oxidized with K2Cr202 to acetate for analysis. Similarly, Cheung and Walsh (71) examined the conversion of D-serine to pyruvate with... 

Although not within the remit of this section, it is of interest to note that threonine is phosphorylated on activation of the phosphorylase from Sac-charomyces cerevisiae. The sequences of amino-acids around this site and around pyridoxal 5 -phosphate were determined the latter, but not the former, sequence is closely homologous to that found in vertebrate phosphorylases. 

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