Clarence Slaughter

Enzymatic synthesis of homocysteine or methionine directly from o-succinyl-homoserine

The bacterial biosynthesis of methionine has previously been shown to involved formation of cystathionine from cysteine and O-succinyl-hormoserine, catalyzed by cystathionine γ-synthase, followed by cleavage of cystathionine to yield homocysteine. This report presents evidence that hydrogen sulfide can replace cysteine as a substrate for cystathionine γ-synthase, yielding homocysteine directly. The maximum velocity of homocysteine formation is about half that of cystathionine formation, but the Km for hydrogen sulfide is 50 times higher than that for cysteine. The leakiness of Salmonella mutants blocked in the conversion of cystathionine to homocysteine suggests that direct synthesis of homocysteine from H2S may take place in the cell, although only to a limited extent. Cystathionine γ-synthase has also been shown to catalyze formation of methionine directly from O-saccinyl homoserine and methyl mercaptan. This reaction is presumed to have no role in the utilization of inorganic sulfur compounds for methionine biosynthesis, but may provide an explanation for the ability of S-methyl cysteine to support the growth of methionine auxotrophs of some microorganisms.

Succinic Ester and Amide of Homoserine: Some Spontaneous and Enzymatic Reactions

O-Succinylhomoserine and N-succinylhomoserine have been synthesized. The first is rapidly transformed into the second by alkali. In acid, the second undergoes ring closure to the lactone, rather than the reverse acyl transfer. Neither supports the growth of methionine auxotrophs of Neurospora or Salmonella. However, bacterial extracts rapidly catalyze formation of a compound, chromatographically identical with cystathionine, from cysteine and O-succinylhomoserine. In the absence of cysteine the O-succinylhomoserine is converted to α-ketobutyrate. Both these reactions are absent from the same Salmonella mutant, and therefore are probably catalyzed by a single enzyme which is needed for methionine synthesis. Both reactions require pyridoxal phosphate. N-succinylhomoserine does not undergo either reaction.

The derepression and function of enzymes of reverse trans-sulfuration in neurospora

Abstract Sulfur transfer from homocysteine to cysteine is mediated by 2 enzymes, cystathionine β-synthase and γ-cystathionase, which are present in animals and fungi, but absent from bacterial species so far examined. It has now been found that when nutrient sulfur limits the growth of Neurospora, the level of γ-cystathionase is increased 30-fold. Derepression is not coordinate, since the level of cystathionine β-synthase is not changed. β-Cystathionase is also unaffected. Non-coordinate derepression suggests that γ-cystathionase might have a more general function in mobilizing sulfur; the enzyme is shown able to rapidly decompose a wide variety of β- or γ-substituted amino acids besides cystathionine. The derepression was shown not to be brought about by starvation in general, and may therefore result from depletion of a low molecular weight, sulfur-containing corepressor

[195e] γ-Cystathionase (Neurospora)

Publisher Summary This chapter discusses the methods of preparation of γ-Cystathionase ( Neurospora ). This pyridoxal-P enzyme is functionally very similar to the comparable rat liver enzyme, though unlike the latter it has not yet been purified to homogeneity. The enzyme is undoubtedly widely distributed among fungi; it has been shown to be present in Saccharomyces as well as Neurospora , and it is absent from some bacterial species. The enzyme is assayed by measuring the rate of formation of either α-ketoacid or mercaptoamino acid. The latter assay is suitable for crude extracts. An aryl disulfide is incorporated into the reaction mixture, and the amount of colored aryl mercaptan produced by spontaneous disulfide interchange is measured continuously in a spectrophotometer. Removal of DPNH oxidase during purification allows an alternative assay for the rate of formation of α-ketoacid, utilizing lactic dehydrogenase. The latter can also be used to determine the proportions of pyruvate and α-ketobutyrate present in a mixture.