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Rat liver gamma-cystathionase has been purified to homogeneity (verified by SDS electrophoresis and ultracentrifugation). The secondary and tertiary structures of the enzyme were studied by circular dichroism spectra. Our studies revealed that the holoenzyme molecule comprises approximately 22% of alpha-helices, 14% of beta-structure, 14% of beta-bends, and 50% of unordered structure. Conformational alterations of the enzyme molecule resulting from enzyme PLP elimination, reduction with sodium borohydride and irreversible inhibition by propargylglycine were examined. The enzyme's secondary structure was shown to be stable whereas the tertiary structure is labile. Saturation with PLP maintains the enzyme's optimal (catalytically active) tridimensional structure. Sodium dodecylsulfate alters its secondary (the amount of alpha-helix being raised to 34%) and tertiary structures.

Cystathionine gamma-lyase (EC 4.4.1.1) is widely distributed in actinomycetes, e.g. genera Streptomyces, Micromonospora, Micropolyspora, Mycobacterium, Nocardia, Streptosporangium, and Streptoverticillium. The enzyme was purified from Streptomyces phaeochromogenes (IFO 3105) in nine steps. After the last steps, the enzyme appeared to be homogenous by the criteria of polyacrylamide gel electrophoresis, analytical centrifugation, and double diffusion in agarose. The enzyme crystallized in the apo form with the addition of ammonium sulfate. The enzyme has a molecular weight of about 166,000 and consists of four subunits identical in molecular weight. The enzyme exhibits absorption maxima at 278 and 421 nm and contains 4 mol of pyridoxal 5'-phosphate/mol of enzyme. L-Cystathionine, L-homoserine, DL-lanthionine, L-djenkolic acid, and L-cystine are cleaved as preferred substrates by the Streptomyces enzyme. The alpha, beta-elimination reaction of L-cystathionine is also catalyzed by the enzyme at a ratio of about one-seventh of the alpha, gamma-elimination reaction. Cystathionine beta-synthase (EC 4.2.1.22) and cystathionine gamma-synthase (EC 4.2.99.9) activities were also detected in crude extracts of S. phaeochromogenes, but cystathionine beta-lyase (EC 4.4.1.8) was not. Consequently, the reverse transsulfuration pathway in actinomycetes may be similar to that in yeast and molds.

A procedure is described for the resolution of beta-cyanoalanine synthase (E.C.4.4.1.9) from blue lupine seedlings. It includes total inhibition of the enzyme the hydroxylamine (10(-2) M) followed by separation of coenzyme oxime from the protein on Sephadex G-25. Conditions for maximal apoenzyme stabilization and reactivation (nature, concentration and pH of the buffer, coenzyme concentration, etc.) were studied. The extent of apoenzyme reactivation by pyridoxal phosphate is found not to depend on the nature of the buffer (pH within the range from 7,1 to 8,8) in the preincubation medium. The interaction were investigated of cyanoalanine aposynthase with pyridoxal phosphate analogues substituted in positions 2,3,4,5 and 6 of the pyridine ring. Only 6-methyl-, 2-nor- and 5'-methyl-pyridoxal phosphate were found to activate apoenzyme to the extention 100, 56 and 31% respectively under the assay conditions. The other analogues tested do not activate apoenzyme, but they mostly interact in the enzyme's active site, competitively inhibiting the binding of pyridoxal phosphate. Ki values were determined for some analogues (according to Dixon). It is found that slight changes in structure of the coenzyme molecule markedly decrease of affinity of the analogue to cyanoalanine aposynthase. As compared with other pyridoxal-phosphate-containing enzymes, this synthase, like serine sulphhydrase and cystationine synthase, has more stringent requirement as to coenzyme structure.