Pyridoxal phosphate serine dehydrase

There is a general requirement for pyridoxal-5-phosphate (24, 25, 27, 44) although not all of the activity lost on dialysis is restored by adding the cofactor. This requirement explains the inhibition by hydroxylamine and hydrazine (24, 25). The reaction is a typical pyridoxal-5-phosphate catalyzed a,/ -elimination with a mechanism similar to serine dehydrase and cysteine desulfhydrase (45). The coenzyme is probably bound as a Schiff base with an amino group of the enzyme since there is an absorption maximum at 415 nm in solutions of the purified garlic enzyme (40). The inhibition by L-cysteine is presumably caused by formation of a thiazolidine with the coenzyme (46). Added pyridoxal-5-phosphate also combines directly with the substrate. The dissociation constant for the complex is about 5 X lO M. When this is taken into account, the dissociation constant of the holoenzyme can be shown to be about 5 X 10 M (47). The higher enzyme activity in pyrophosphate buflFer than in Tris or phosphate may be explained by pyrophosphate chelation of metal ions which otherwise form tighter complexes with the substrate and coenzyme (47). This decreases the availability of added coenzyme. 

Serine and threonine dehydrases. Serine and threonine are not substrates in transamination reactions. Their amino groups are removed by the pyridoxal phosphate-requiring hepatic enzymes serine dehydratase and threonine dehydratase. The carbon skeleton products of these reactions are pyruvate and a-keto-butyrate, respectively. 

Mammalian tissues contain enzymes that catalyze the nonoxidative deamination of serine, threonine, and homoserine. Since the postulated reaction mechanism involves a dehydration before the deamination, these enzymes are called dehydrases. L-Serine, L-threonine, and L-homoserine dehydrases have been partially purified and all are specific for the L-amino acid. Serine and threonine dehydrases require pyridoxal phosphate, ATP, and glutathione for activity. Pyridoxal phosphate requires the homoserine enzyme, but the need for ATP and glutathione has not been demonstrated. The reaction is likely to involve the formation of a Schiff base. The homoserine dehydrase has been... 

D-Serine dehydrase from both E. coli and Neurospora required pyridoxal phosphate as a coenzyme. In contrast to L-serine dehydrase, threonine is attacked very poorly by the D-enz3une. Magnesium ions had no activating affect on the E. coli enzyme, but it partially reversed the inhibition caused by the metal-binding reagents, phosphate, citrate, cysteine, cyanide, and 8-hydroxyquinoline of the Neurospora enzyme. ... 

Umbarger and Brown 233) have observed L- and D-threonine dehy-drases in E. coli which are stimulated by pyridoxal phosphate. In a continuation of this work 234) they have presented evidence that E. coli extracts contain two distinct L-threonine dehydrases. One of the dehydrases is the L-threonine dehydrase of Wood and Gunsalus and requires pyridoxal phosphate, AMP, and glutathione. It was found to be an adaptive enzyme, and was active against L-serine. The second dehydrase is present in extracts of wild-type E. coli, but not in mutants which are unable to convert threonine to a-ketobutyrate as a step in isoleucine synthe. It requires only pyridoxal-5-phosphate and is inhibited by isoleucine. On the basis of kinetic studies it is proposed that the Wood-Gunsalus dehydrase combines with one molecule of substrate, whereas the other enzyme combines with two. L-Serine is a substrate for both enz3unes. Evidence is presented for a third deaminase in E. coli cells which is serine-specific. 

The preparation in a cell-free form of a D-serine dehydrase from E. coli which requires pyridoxal phosphate has been described by Metzler and Snell 218). This enzyme is readily separated from the L-serine (and threonine) dehydrase of Wood and Gunsalus 216). Unlike the latter enzyme, the D-serine dehydrase does not require AMP or glutathione. DL-Threonine was slowly deaminated by the system. 

Both isomers of both serine and threonine are deaminated by Neuro-spora extracts 225-228). The enzymes involved have been purified by Yanofsky and his associates 225-228). A specific D-serine and D-threonine dehydrase has been purified thirty-five- to fortyfold from this mold 2f ). An absolute requirement for pyridoxal phosphate was demonstrated. No requirement for AMP or glutathione could be demonstrated. Some indication of a metal requirement was observed. The preparation was not active with the li-isomers of serine and threonine or DL-homoserine and DL-homo-cysteine. The rate of deamination of D-threonine is very slow compared to that with L-serine. Activity was observed with D-glutamic acid and D-as-partic acid. Since other D-amino acids were not deaminated by the preparation, these results could not be due to a contamination with D-amino acid oxidase. Furthermore, when either of these amino acids was incubated in the presence of D-serine, the keto acid production was a summation of that for each substrate alone. Pyridoxal phosphate had no effect on keto acid formation from the dicarboxylic amino acids. It is of interest that D-amino acid oxidase of Neurospora does not attack D-serine or D-threonine (77). 

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