Subject pyridoxal-5 -phosphate

W2. Wachstein, M., Kellner, J. D., and Oritz, J. M., Pyridoxal phosphate in plasma and leukocytes of normal and pregnant subjects following Be load tests. Froc. Soc. Exptl. Biol. Med. 103, 350-353 (1960). 

As indicated in Section 6.3.3 and Table 6.2 the key control step is mediated by glycogen phosphorylase, a homodimeric enzyme which requires vitamin B6 (pyridoxal phosphate) for maximum activity, and like glycogen synthase (Section 6.2) is subject to both allosteric modulation and covalent modification. 

Structures of vitamin B6 derivatives and the bonds cleaved or formed by the action of pyridoxal phosphate (a). The reactive part of the coenzyme is shown in red in (a). The bonds shown in red in (d) are the types of bonds in substrates that are subject to cleavage. 

This reaction is catalyzed by the enzyme ALA synthase (Fig. 2d) which requires the coenzyme pyridoxal phosphate (see Topic M2) and is located in the mitochondria of eukaryotes. This committed step in the pathway is subject to regulation. The synthesis of ALA synthase is feedback-inhibited by heme. 

In subjects with hypophosphatasia, the rare genetic lack of extracellular alkaline phosphatase, plasma concentrations of pyridoxal phosphate are very much higher than normal (up to 4 /rmol per L, compared with a normal range of about 100 nmol per L), and intracellular concentrations of pyridoxal phosphate are lower than normal (Narisawa et al., 2001). 

Free pyridoxal either leaves the cells or is oxidized to 4-pyridoxic acid by aldehyde dehydrogenase (which is present in all tissues) and also by hepatic and renal aldehyde oxidases. 4-Pyridoxic acid is actively secreted by the renal tubules, so measurement of the plasma concentration provides an index of renal function (Coburn et al., 2002). There is some evidence that oxidation to 4-pyridoxic acid increases with increasing age in elderly people, the plasma concentration of pyridoxal phosphate is lower, and that of 4-pyridoxic acid higher, than in younger subjects even when there is no evidence of impaired renal function (Bates et al., 1999b). Small amounts of pyridoxal and pyridox-amine are also excreted in the urine, although much of the active vitamin Be that is filtered in the glomerulus is reabsorbed in the kidney tubules. 

The histidine decarboxylase activity of tissues can be raised or lowered by changing the hormonal state of the animal or by subjecting it to certain stressful stimuli. Administration of thyroid hormones to rats produces a marked increase in the specific histidine decarboxylase activity of the glandular mucosa of the stomach " , while the activity of the nonspecific enzyme in the liver is lowered . Studies of the action of thyroid hormones on other pyridoxal-dependent enzymes in rat liver suggest that, in this organ at least, these changes arise from corresponding alterations in both pyridoxal phosphate and apo-enzyme synthesis . 

Figure 11.13 Reactions at a-carbon of a-amino acids catalyzed by pyridoxal enzymes All three substituents at C are subject to labilization in the three types of a-carbon reactions. The hydrogen is labilized in recemization reactions, the amino group is labUized in the transamination and the carboxyl group is labilized in decarboxylation. a-Amino acid condenses with pyridoxal phosphate to yield pyridoxylidene imino acid (an aldimine). The common intermediate, aldimine and distinct ketimines leading to the production of oxo-acid (in transamination), amino acid (in racemization) and amine (in decarboxylation) are shown. The catalytic acid (H-A-) and base (-B ) are symbolic both can be from the same residue such as Lys258 in aspartate aminotransferase.

Each monomer contains one pyridoxal phosphate and a highly conserved primary structure in the vicinity of the cofactor binding site. The enzymes follow the same catalytic mechanism, a rapid equilibrium random Bi Bi mechanism. However, unlike animal phosphorylases, the microbial and plant enzymes are not subjected to covalent or allosteric control. Within the group of higher plant phosphorylases two types of enzyme have been distinguished which differ in monomer size, peptide pattern, glucan specificity, and intracellular location (1-3). Based on immunochemical studies on leaf tissues (4-5), one enzyme form has been localized in the cytosol whereas the other one resides in the chloroplast. Thus, the two plant phosphorylase types represent non-interconvertible proteins which presumably have entirely different metabolic functions. 

Previous investigations of this subject led to the misleading results that oxygen was consumed and CO2 produced in the reaction also that riboflavin and DPN, as well as pyridoxal phosphate, were required for maximal activation. The confusion resulted from the fact that oxidation of pyruvate was being measured in the crude bacterial preparations as well as the decomposition of tryptophan to indole. For literature, see . 

K Dalery, S Lussier-Cacan, J SeUmb, J Davignon, Y Latour, J Genest. Homocysteine and coronary artery disease in French Canadian subjects relation with vitamins B-12, B-6, pyridoxal phosphate and folate. Am J Cardiol 75 1107-1111, 1995. 

Pyridoxal phosphate was demonstrated to be required as a coenzyme by dissociating it from the apoenzyme. This causes a loss of enzyme activity which can be restored by recombining the apoenzyme with pyridoxal phosphate. The optimum activity is at pH 8.3 and the enzyme requires free sulfhydryl groups for activity. The serine deaminase activity of the enz3rme is strongly inhibited by homocysteine and by cystathionine. No synthetic product appears to be formed with cysteine. The Km of the enzyme for serine is 8.1 X 10 Af. Knowledge of the mechanisms of the reactions catalyzed by pyridoxal phosphate-dependent enzymes has been advanced by model experiments with pyridoxal, metal ions, and amino acids. This subject has been reviewed recently by Snell (61). 

A number of studies have shown that between 10 and 20% of the apparently healthy population have low plasma concentrations of pyridoxal phosphate or abnormal erythrocyte transaminase activation coefficient, suggesting vitamin Bg inadequacy or deficiency. In most studies, only one of these indices of vitamin Bg nutritional status has been assessed. Where both have been assessed, while each shows some 10% of the population apparently inadequately provided with vitamin Bg, few of the subjects show inadequacy by both criteria. 

Although, owing to the wide distribution of vitamin Bg in nature, clinical deficiency symptoms are seldom observed, there is little doubt that pyridoxine is essential in human nutrition. Pyridoxine is absorbed from the gastrointestinal tract and is converted to the active form pyri-doxal phosphate. Absorption is decreased in gastrointestinal diseases and also in subjects taking isoniazid (3). It is excreted in the urine as 4-pyridoxic acid (2). The metabolism of vitamin Bg in human beings has been investigated (56). 

These results show that the phosphate and aldehyde moieties of pyridoxal-5-phosphate and its analogues are required for antagonist activity at P2X Punnoceptors in rabbit vas deferens. Among this series of compounds, PPADS was found to be the most interesting P2-antagonist, and it was therefore the subject of a more detailed pharmacological investigation. 

The catabolic pathway of vitamin Bg (1) is probably the best studied. In animals (including humans) 4-pyridoxic acid (4) is the primary catabolic product of vitamin Bg, found in urine. It is formed by the oxidation of pyridoxal (2) by a nonspecific flavin adenine dinucleotide (FAD)-dependent aldehyde oxidase. The catalytically active form of vitamin Bg, pyridoxal-5 -phosphate, PLP (17), does not undergo similar oxidation to form 4. The various forms of vitamin Bg (1, 2, 15) and their respective phosphate esters (16, 17) are readily enzymatically interconvertible. Further degradation of 4 is unlikely in humans as the subjects administered with doses of 4 were found to excrete it quantitatively. Estimation of 4 in urine serves as a nutritional marker, as lower than normal amounts of 4 is indicative of vitamin Bg deficiency. ... 

A more specific type of chemical assay is based on enzymatic measurement of vitamin co-enzyme activity. This approach is designed to detect a vitamin deficiency in tissues, and is only feasible for those vitamins that serve as co-enzymes. For instance, thiamin depletion in a subject can be diagnosed by measuring the transketolase activity in red blood cells with and without the addition of thiamin pyrophosphate (TPP) in vitro. If TPP increases the activity by more than a given amount, thiamin deficiency is indicated. Similarly, a subnormal level of riboflavin is indicated in tissues if the activity of erythrocyte glutathione reductase is increased after the addition of flavin adenine dinucleotide (FAD). Erythrocyte transaminase activation by pyridoxal-5 -phosphate (PLP) can be measured to establish a deficiency of vitamin B . 

The reactions of amino acids, catalysed by enzymes requiring a derivative of vitamin Bg (Figure 5a) as cofactor, have been the subject of a number of mechanistic studies. Enzymes catalysing decarboxylation, racemisation, dehydration (of serine or threonine) or desulphy-dration (of cysteine) require pyridoxal-5 -phosphate as cofactor (Figure 5b), and are dealt with in later sections. Enzymes catalysing transamination, on the other hand, require either pyridoxal-5 -phosphate or pyridoxamine-5 -phosphate (Figure 5c). Snell and coworkers showed that the reactions of amino acids normally... 

To better understand the mode of action of pyridoxal-P, let us examine in detail equation 7-3 the conversion of homosereine phosphate to threonine. It is an elimination-hydration transformation. The first process (Fig. 7.10) involves an aldimine ketimime tautomerization which is subjected to general-acid catalysis intramolecularly by the hydroxyl group, followed by the slow breaking of a C—H bond. The latter is the rate-determining step. 

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