Pyridoxal phosphate, coenzyme cofactor

Pyridoxal phosphate is a coenzyme for many enzymes involved in amino acid metabolism, especially in transamination and decarboxylation. It is also the cofactor of glycogen phosphorylase, where the phosphate group is catalytically important. In addition, vitamin Bg is important in steroid hormone action where it removes the hormone-receptor complex from DNA binding, terminating the action of the hormones. In vitamin Bg deficiency, this results in increased sensitivity to the actions of low concentrations of estrogens, androgens, cortisol, and vitamin D. 

We have just noted the role that pyridoxal phosphate plays as a coenzyme (cofactor) in transamination reactions (see section 15.6). Pyridoxal 5 -phosphate (PLP) is crucial to a number of biochemical reactions. PLP, together with a number of closely related materials that are readily converted into PLP, e.g. pyridoxal, pyridoxine and pyridoxamine, are collectively known as vitamin Bg, which is essential for good health. 

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. 

This enzyme [EC 2.3.1.37] catalyzes the reaction of suc-cinyl-CoA with glycine to yield 5-aminolevulinate, coenzyme A, and carbon dioxide. Pyridoxal phosphate is used as a cofactor in this reaction. In mammals, the enzyme isolated from erythrocytes is genetically distinct from that in other tissues. 

The first examples of mechanism must be divided into two principal classes the chemistry of enzymes that require coenzymes, and that of enzymes without cofactors. The first class includes the enzymes of amino-acid metabolism that use pyridoxal phosphate, the oxidation-reduction enzymes that require nicotinamide adenine dinucleotides for activity, and enzymes that require thiamin or biotin. The second class includes the serine esterases and peptidases, some enzymes of sugar metabolism, enzymes that function by way of enamines as intermediates, and ribonuclease. An understanding of the mechanisms for all of these was well underway, although not completed, before 1963. 

In addition to serving as structural motifs, enols and enolates are involved in diverse biological processes. Several enol/enolate intermediates have been proposed to be involved in glycolysis (Section IV.A), wherein c/ -enediol 21 is proposed to be an intermediate in the catalytic mechanism of phosphohexose isomerase and an enol-containing enamine intermediate (22) has been proposed in the catalytic pathway of class I aldolase. In the case of glucose-fructose (aldose-ketose) isomerization, removal of the proton on Cl-OH produces the aldose while deprotonation of C2-OH yields the ketose, which is accompanied by protonation at the C2 and Cl positions, respectively. There are several cofactors that are involved in various biological reactions, such as NAD(H)/NADP(H) in redox reaction and coenzyme A in group transfer reactions. Pyridoxal phosphate (PLP, 23) is a widely distributed enzyme cofactor involved in the formation of a-keto acids, L/D-amino... 

The chemistry of the cofactors has provided a fertile area of overlap between organic chemistry and biochemistry, and the organic chemistry of the cofactors is now a thoroughly studied area. In contrast, the chemistry of cofactor biosynthesis is stiU relatively underdeveloped. In this review the biosynthesis of nicotinamide adenine dinucleotide, riboflavin, folate, molyb-dopterin, thiamin, biotin, Upoic acid, pantothenic acid, coenzyme A, S-adenosylmethionine, pyridoxal phosphate, ubiquinone and menaquinone in E. coli will be described with a focus on unsolved mechanistic problems. 

This chapter describes model studies of hydride transfer entirely with respect to nicotinamide coenzymes, flavin coenzymes and quinone coenzymes. Other coenzymes/cofactors may be alluded to but are not reviewed in detail. Some coenzymes involved either in hydride transfer or the transfer of other hydrogen species have been treated elsewhere in these volumes (thiamin diphosphate is treated by Hiibner et al., pyridoxal phosphate by Spies and Toney, folic acid by Benkovic... 

E. W. Miles, Pyridoxal phosphate enzymes catalyzing fl-elimination and 3-replacement reactions, in Pyridoxal Phosphate and Derivatives, Vol. I in the series Coenzymes and Cofactors, (eds. D. Dolphin, R. Poulson, and O. Avramovic), John Wiley and Sons, New York, 1986, pp. 253-310. 

In contrast to our understanding of the biosynthesis of cofactors, relatively little is known about cofactor degradation. Some previous research has been carried out to identify intermediates on these catabolic pathways, but very little information is available on the genes involved and on the enzymol-ogy. In this chapter we summarize our current understanding of the pyridoxal phosphate, riboflavin, heme, thiamin, biotin, nicotinamide adenine dinucleotide (NAD), folate, lipoate, and coenzyme A catabolic pathways in all life-forms and discuss mechanistic aspects of the most interesting catabolic reactions. 

Conversion of serine to glycine. This reaction requires tetrahydrofolate as an acceptor of a methylene group from Ser and utilizes pyridoxal phosphate as a cofactor. It results in the formation of 5,10-CH PteGlu, an essential coenzyme for the synthesis of thymidylate. 

Amino acid metabolism requires the participation of three important cofactors. Pyridoxal phosphate is the quintessential coenzyme of amino acid metabolism (see Chapter 38). All amino acid reactions requiring pyridoxal phosphate occur with the amino group of the amino acid covalently bound to the aldehyde carbon of the coenzyme (Fig. 39.3). The pyridoxal phosphate then pulls electrons away from the bonds around the a-carbon. The result is transamination, deamination, decarboxylation, P-elimination, racemization, and -elimination, depending on which enzyme and amino acid are involved. 

The transamination of P-aminoisobutyrate to form methylmalonate semialdehyde requires pyridoxal phosphate as a cofactor. This reaction is similar to the conversion of ornithine to glutamate y-semialdehyde. Then NAD+ serves as an electron acceptor for the oxidation of methylmalonate semialdehyde to methylmalonate. The conversion of methylmalonate to methylmalonyl CoA requires coenzyme A. The final reaction, in which methylmalonyl CoA is converted to succinyl CoA, is catalyzed by methylmalonyl CoA mutase, an enzyme that contains a derivative of vitamin B12 as its coenzyme. 

Pyridoxal phosphate is a cofactor required for several reactions involving amino acid interconversion. Its requirement is increased in relation to the amount of protein in the diet. Some of the deficiency symptoms can be readily correlated with their coenzyme function. Thiamin pyrophosphate is a cofactor for pyruvate dehydrogenase, the activity of which is decreased in the brain as a result of deficiency. Pantothenic acid is required not only for... 

All fransamination reactions are reversible and need pyridoxal phosphate (PLP) as the coenzyme. The cofactor (PLP) is covalently bonded to the amino group of an active site lysine, forming an internal aldimine. 

It is interesting that enzyme reactions of primary and secondary metabolism show the same stereospecificity even if there is no discernible advantage of one mode of stereochemical reaction over the other. Examples are the pyridoxal phosphate-dependent enzymes (C 5) which all have the same stereochemistry of the coenzyme-substrate complex, though an alternative conformer formed by 180° rotation could operate equally well. Hence all pyridoxal phosphate-dependent enzymes obviously evolved from a common ancestral protein in which an arbitrary choice between the two faces of the cofactor was made. 

Vitamin Bg (pyridoxine also known as pyridoxol, 118) is an essential growth factor in the diet of many organisms and animals. It forms part of a coenzyme (pyridoxylphosphate) and it is a cofactor for a class of enzymes known as transaminases. A transaminase or an aminotransferase is an enzyme that catalyzes a type of reaction between an amino acid and an a-keto acid. The presence of elevated transaminase levels can be an indicator of liver damage. Vitamin Bg has both an aldehyde form (pyridoxal, 119) and an amine form (pyridoxamine, 120), and it is known that pyridoxal phosphate is a carrier of amino groups and sometimes amino acids. ... 

In biochemical parlance, these systems are called mutases, or sometimes isomerases. When Z = OH and Y = OH or NHj, the product eliminates an aldehyde and either HjO or NH3 so that the process is irreversible. Such systems sometimes are referred to as eliminases, dehydrases or ammonia-lyases. Examples of these various types of systems are shown by the first five examples in Figure 8.3, where (CoA)—S represents coenzyme A. It should be noted that the amino mutases, such as ornithine amino mutase, also require pyridoxal phosphate as a cofactor. 

The existence of enzymes in microorganisms which catalyze the interconversion of D- and L-amino acids is of considerable interest, since the intramolecular transfer of an amino group is apparently involved. The term racemase has been proposed for such enzymes. Two racemases have been reported. Alanine racemase has been shown to be present in a large number of microorganisms and has been partially purified from extracts of S. faecalis. Glutamic acid racemase has been demonstrated in acetone powders of Lactobacillus arabinosus. Both enzymes catalyze the interconversion of the n- and l- forms of their respective substrates. Alanine racemase requires pyridoxal phosphate as coenzyme. Pyri-doxamine phosphate under the conditions employed was not active. Glutamic acid racemase was found not to be affected by the addition of pyridoxal phosphate. However, further studies with purified preparations are necessary before pyridoxal phosphate can be excluded as cofactor for the glutamic acid racemase. Examination of animal tissues under conditions favorable for the demonstration of bacterial alanine racemase failed to reveal any activity. 

The enzyme which catalyzes the formation of tryptophan from indole and serine has been named tryptophan desmolase. A cell-free preparation has been obtained from Neurospora mycelium which catalyzes this reaction. Pyridoxal phosphate has been found to be a necessary cofactor. The enzyme has been partially purified by Yanofsky. He found the optimum activity to be at pH 7.8, and confirmed the necessity of pyridoxal phosphate as a coenzyme. Effective inhibitors are Co++, Zn++, CN, hydroxylamine, and tryptophan. 

Pyridoxal phosphate has been established as a coenzyme in two reactions involving tryptophan. An enzyme has been isolated from Neurospora which catalyzes a synthesis of tryptophane from serine and indole. This reaction requires pyridoxal phosphate. An enzyme has been isolated from E. coli which causes the decomposition of tryptophan to pyruvic acid, indole, and ammonia here too pyridoxal phosphate is a necessary cofactor. 

M Ebadi. Catabolic pathways of pyridoxal phosphate and derivatives. In D Dolphin, R Poulson, O Avramovic, eds. Coenzymes and Cofactors, Vol. 1. Vitamin Be—Pyridoxal Phosphate Chemical, Biochemical and Medical Aspects, part B. New York John Wiley Sons, 1986, pp 449-476. 

One important subgroup of the lyases are the decarboxylases. The decarboxylation of amino acids is assisted by pyridoxal phosphate as a prosthetic group, whereas in the decarboxylation of pyruvate to acetaldehyde, thiamine pyrophosphate (TPP) plays that role. Oxidative decarboxylation, lastly, depends on the cooperation of no fewer than five cofactors thiamine pyrophosphate, lipoic acid, coenzyme A, flavin-adenine dinucleotide, and nicotinamide-adenine dinucleotide. 

---