Protein reaction with pyridoxal phosphate

A few years later, in 1953, the versatility of pyridoxal phosphate was illustrated by Snell and his collaborators who found many of the enzyme reactions in which pyridoxal phosphate is a coenzyme could be catalyzed non-enzymically if the substrates were gently heated with pyridoxal phosphate (or free pydridoxal) in the presence of di- or tri-valent metal ions, including Cu2+, Fe3+, and Al3+. Most transaminases however are not metal proteins and a rather different complex is formed in the presence of the apoprotein. 

Proteins that have tightly bound cofactors, such as heme proteins, photosynthetic reaction centers and antenna proteins, flavoproteins, and pyridoxal phosphate- and NAD-dependent enzymes, provide a variety of chromophores which have absorption bands in the visible and UV region. The CD bands associated with the chromophoric groups are frequently quite intense, despite the fact that the isolated chromophores are achiral in many cases, and therefore have no CD, or are separated from the nearest chiral center by several bonds about which relatively free rotation can occur, and therefore have only weak CD. The extrinsic or induced CD observed in the visible and near-UV spectra of the proteins can provide useful information about the conformation and/or environment of the bound chromophore, which usually plays a critical role in the function of the protein. 

Fig. 2. Tentative scheme for the conversion of glycine to serine associated with the inside of the inner membrane and matrix of plant mitochondria. E, Ej, E3, and E4 are regarded as enzyme proteins with pyridoxal phosphate, lipoyl, possibly tetrahydrofolate and FAD as prosthetic groups or cofactors. The scheme incorporates published information for similar systems in microorganisms, liver mitochondria, and plants and shows the three phosphorylations of ADP to ATP considered to be associated with the reaction in plants.

Extensive research work has gone into modification of proteins, not for commercial applications but for academic reasons. Thus, for instance, Frances et al. developed a new reaction that introduces single reactive ketones or aldehydes at the N-terminal groups of protein when the proteins are mixed with pyridoxal phosphate [44]. The researchers also developed a palladium-catalyzed allylic alkylation that attaches long lipid tails to proteins, a process that can be used to customize the solubihty of enzymes, antibodies, viral capsids, and other proteins. 

This pyridoxal-phosphate-dependent enzyme [EC 2.1.2.5], also known as glutamate formyltransferase, catalyzes the reaction of 5-formiminotetrahydrofolate with L-glutamate to produce tetrahydrofolate and A-formim-ino-L-glutamate. The enzyme will additionally catalyze the transfer of the formyl moiety from 5-formyltetrahy-drofolate to L-glutamate. This protein occurs in eukaryotes as a bifunctional enzyme also having a formiminote-trahydrofolate cyclodeaminase activity [EC 4.3.1.4]. 

Fig. 5 Catalytic mechanism of glycogen phosphorylases. The reaction scheme accounts for the reversibility of phosphorolysis of oligosaccharides (R) in the presence of orthophosphate (upper half) and primer-dependent synthesis in the presence of glucose-l-phosphate (lower half). PL enzyme-bound pyridoxal BH-f a general base contributed by the enzyme protein. Reprinted with permission from [109]. Copyright 1990 American Chemical Society...

Aminomutases. The enzymes L-p-lysine mutase (which is also D-a-lysine mutase) and D-omithine mutase catalyze the transfer of an co-amino group to an adjacent carbon atom (Table 16-1). Two proteins are needed for the reaction pyridoxal phosphate is required and is apparently directly involved in the amino group migration. In the P-lysine mutase the 6-amino group of L-P-lysine replaces the pro-S hydrogen at C-5 but with inversion at C-5 to yield (3S, 5S)-... 

Pyridoxal phosphate is the coenzyme in a large number of amino acid reactions. At this point it is convenient to consider together 1,he mechanism of those pyridoxal-dependent reactions concerned with aromatic amino acids. The reactions concerned are (1) keto acid formation (e.g., from kynurenine, above), 2) decarboxylation (e.g., of 5-hydroxytrypto-phan to 5-hydroxytryptamine, p. 106), (3) scission of the side claain (e.g., 3-tyrosinase, p. 78 tryptophanase, p. 110 and kynureninase, above), and 4) synthesis (e.g., of tryptophan from indole and serine, p. 40). Many workers have considered the mechanism of one or more of these reactions (e.g., 24, 216, 361, 595), but a unified theory is primarily due to Snell and his colleagues (summarized in 593). Snell s experiments have been carried out largely in vitro, and it should be emphasized that in vivo it is the enzyme protein which probably directs the electromeric changes. 

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... 

H) 4-Amino-4-deoxychorismate lyase (EC 4.1.3.38) PabC protein (Figure 2, 1) catalyzes the elimination of pyruvate (21) from 4-amino-4-deoxychorismate (20). The enzyme utilizes pyridoxal phosphate as a cofactor. The reaction mechanism shown in Figure 4 implies that pyridoxal phosphate forms a Schiff base (23) with the substrate, 4-amino-4-deoxychorismate. Abstraction of a proton from C-4 of the amino-4-deoxychorismate moiety is believed to result in a shift of the imine bond. Pyruvate can then be eliminated from the Schiff base form 23, and the pyridoxal phosphate moiety can be transferred back to a lysine residue (specifically, lysine 159 of coli PabC protein) under liberation of 4-amino benzoate (14) as shown by X-ray crystallography (Figure 4). ... 

Primary amines (such as the e-amino group of lysine) react readily with aldehydes, often forming Schiff bases which can be stabilized by reduction with NaBH4. Such a reaction forms the basis of the widespread use of pyridoxal phosphate to modify chemically the lysine residues of proteins. In recent years, in order to increase the specificity of labeling, aldehydes have been incorporated into more complex compounds which mimic the structures of the substrate or cofactor of the enzyme. 

---