Pyridoxal phosphate-containing enzymes

Canaline is a potent inhibitor of all seven pyridoxal phosphate-containing enzymes studied by Rahiala et (27) but it lacks adverse effects on three ornithine-utiTTzing enzymes lacking a Bg cofactor. Finally, in jack bean, Canavalia ensiformis, ornithine carbamoyl transferase can form 0-ureido-L-homoserine from canaline and carbamoyl phosphate as it does citrulline from ornithine and carbamoyl phosphate. Nevertheless, neither compound inhibited formation of the reaction products (31). 

The subsequent cleavage of cystathionine to yield cysteine, a-ketobutyrate and NH4+ is catalyzed by y-cystathionase, a pyridoxal-phosphate-containing enzyme. This transsulfura-tion pathway is one of the routes used for methionine catabolism. 

Labilization of bonds of an amino acid bound to pyridoxal phosphate-containing enzymes. Given the appropriate apoenzyme, any atom or group on the carbon atom proximal to the Schiff base can be cleaved. 

Structures of compounds that inhibit pyridoxal phosphate-containing enzymes. 

Canaline is the product of the hydrolytic cleavage of canavanine with the simultaneous formation of urea. Canaline is an ornithine analogue which also shows neurotoxicity in the adult sexta where it adversely affects central nervous system functions (jj ). It also is a potent inhibitor of vitamin B -containing enzymes (20-22). It forms a stable Schiff base with the pyridoxal phosphate moiety of the enzyme and drastically curtails enzymatic activity. Pyridoxal phosphate-containing enzymes are vital to insects because they function in many essential transamination and decarboxylation reactions. Ornithine is an important metabolic precursor for insect production of glutamic acid and proline (23). 

All of the nitrogen in heme is derived from glycine and all of the carbons are derived from succinate and glycine. Thus, the process by which heme is synthesized is also called the succinate-glycine pathway. The first step in the process is catalyzed by a pyridoxal phosphate-containing enzyme, 5-aminolevulinic acid synthetase (ALA synthetase)... 

Orientational dependency of pyridoxal phosphate containing enzyme catalyzed reactions... 

Werle and Pechmann 184) have presented evidence that diamine oxidase from plants could be activated by pyridoxal. The rate of inhibition of enzyme systems by isonicotinic acid hydrazide has been used as a means of detecting the presence of pyridoxal phosphate as a coenzyme, particularly when it is tightly bound to the apoenzyme 205, 206). Davison 166) has shown that the rates of inhibition of an established pyridoxal phosphate containing enzyme, cysteinesulfinic acid decarboxylase 207, 208), and diamine oxidase are similar and that pyridoxal phosphate will reactivate diamine oxidase after inhibition with isonicotinic acid hydrazide. On this basis it has been suggested that pyridoxal phosphate is probably involved as a coenzyme for diamine oxidase. 

Breakdown of glycogen to glucose 1-phosphate requires the presence of phosphate as a cosubstrate and is catalyzed by glycogen phosphorylase, a pyridoxal phosphate-containing enzyme (MW 200,000). The enzyme is exceptional because in contrast to all other enzymes carrying this coenzyme, reduction with sodium borohydiide does not result in r uction of a Schifi -... 

The biosynthesis of tryptophan occurs by condensation of L-serine with indole, this reaction is catalyzed by tryptophan synthetase. The enzyme is a pyridoxal-phosphate containing enzyme which catalyzes nucleophilic p-substitution reactions of amino acids. The p-hydroxyl group of serine is substituted by indole by the action of the enzyme. The reaction is thought to proceed via a ketimine intermediate (27) which undergoes elimination to give an aminoacrylate-pyridoxal phosphate Schiff base (28). Addition at the P-carbon of indole followed by reversal of the process constitutes the enzymatic synthesis of L-tryptophan. 

Diamine Oxidases. Diamine oxidases are enzymes that also oxidize amines to aldehydes. The preferred substates are aliphatic diamines in which the chain length is four (putrescine) or five (cadaverine) carbon atoms. Diamines with carbon chains longer than nine will not serve as substrates but can be oxidized by monoamine oxidases. Secondary and tertiary amines are not metabolized. Diamine oxidases are typically soluble pyridoxal phosphate-containing proteins that also contain copper. They have been found in a number of tissues, including liver, intestine, kidney, and placenta. 

A number of enzymes that catalyze the same reactions as do pyridoxal phosphate-dependent enzymes contain a catalytic pyruvate residue at the amino terminal of the peptide chain. The catalytic mechanism is assumed to be the same as for pyridoxal phosphate-dependent enzymes, except that the proton donor is a glutamate residue rather than lysine. 

Vitamin B6 is one of the most versatile enzyme cofactors. Pyridoxal phosphate-containing proteins are found in each lUB enzyme category except ligases (category 6). [Tong and Davis (1995) reported that 2-amino-3-ketobutyrate-CoA ligase is a pyridoxal phosphate enzyme. However, the... 

The requirement for metal ions in these non-enzymatic transamination reactions suggests that such ions may be involved as catalysts in the enzymatic reactions. However, most of the pyridoxal phosphate dependent enzymes that have been purified to date do not contain metal ions, and addition of the latter to the reaction medium does not increase the rate of enzymatic reactions Metal ions, therefore, appear to fulfill some of the roles in non-enzymatic reactions played by the protein in enzymatic reactions, but the protein is apparently a much more efficient catalyst. 

Muscle phosphorylase is distinct from that of Hver. It is a dimer, each monomer containing 1 mol of pyridoxal phosphate (vitamin Bg). It is present in two forms phos-phoiylase a, which is phosphorylated and active in either the presence or absence of 5 -AMP (its allosteric modifier) and phosphorylase h, which is dephosphorylated and active only in the presence of 5 -AMP. This occurs during exercise when the level of 5 -AMP rises, providing, by this mechanism, fuel for the muscle. Phosphorylase a is the normal physiologically active form of the enzyme. 

The Schiff base can undergo a variety of reactions in addition to transamination, shown in Fig. 6.4 for example, racemization of the amino acid via the a-deprotonated intermediate. Many of these reactions are catalyzed by metal ions and each has its equivalent nonmetallic enzyme reaction, each enzyme containing pyridoxal phosphate as a coenzyme. Many ideas of the mechanism of the action of these enzymes are based on the behavior of the model metal complexes. 

The cases of myoglobin and hemoglobin are not rare. Many enzymes are dependent for their function on the presence of a nonprotein group. For example, cytochrome c also contains a prosthetic group similar, but not identical, to heme, as do a number of other proteins. These are known generically as heme proteins. There is a family of enzymes that contain a flavin group, the flavoproteins. Another family contains pyridoxal phosphate, a derivative of vitamin Be. There are a number of other examples. 

The active form of vitamin Be, pyridoxai phosphate, is the most important coenzyme in the amino acid metabolism (see p. 106). Almost all conversion reactions involving amino acids require pyridoxal phosphate, including transaminations, decarboxylations, dehydrogenations, etc. Glycogen phosphory-lase, the enzyme for glycogen degradation, also contains pyridoxal phosphate as a cofactor. Vitamin Be deficiency is rare. 

Non-pyridoxal Phosphate Dependent. Figure 2 depicts the postulated mechanism for a non-pyridoxal phosphate catal) zed decarboxylation of histidine to histamine involving a pyruvoyl residue instead of pyridoxal -5 - phosphate (20). Histidine decarboxylases from Lactobacillus 30a and a Micrococcus sp. have been shown to contain a covalently bound pyruvoyl residue on the active site. The pyruvoyl group is covalently bound to the amino group of a phenylalanine residue on the enzyme, and is derived from a serine residue (21) of an inactive proenzyme (22). The pyruvoyl residue acts in a manner similar to pyridoxal phosphate in the decarboxylation reaction. 

L-Canaline is an ineffective antimetabolite of L-ornithine since it has little ability to antagonize ornithine-dependent reactions. On the other hand, it forms a covalently bound Schiff-base complex with the pyridoxal phosphate moiety of Bg-containing enzymes. As such it is a potent inhibitor of many decarboxylases and aminotransferases that utilize this vitamin. 

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