Beta-cyano-L~alanine synthase iL-cysteine hydrogen-sulfide-lyase (adding HCN), EC 4.4.1.91 was purified about 4000-fold from blue lupine seedlings. The enzyme was homogeneous on gel electrophoresis and free of contamination by other pyridoxal-P-dependent lyases. The enzyme has a molecular weight of 52,000 and contains 1 mole of pyridoxal-P per mole of protein; its isoelectric point is situated at pH 4.7. Its absorption spectrum has two maxima, at 280 and 410 nm. L-Cysteine is the natural primary (amino acid) substrate; jB-chloro-and ,3-thiocyano-L-alanine can substitute for it. Some small aliphatic thiols can serve (with considerably lower affinity) instead of cyanide as cosubstrates for cyanoalanine synthase. The synthase is refractory to DL-cycloserine and D-penicillamine, potent inhibitors of many pyridoxal-P-dependent enzymes. Cyanoalanine synthase catalyzes slow isotopic a-H exchange in cysteine and in end-product amino acids; the rates of a-H exchange in nonreacted (excess) cysteine are markedly increased in the presence of an adequate cosubstrate; no exchange is observed of H atoms in f3-position. In recent years we made comparative studies of some vitamin-B6-dependent lyases exclusively catalyzing replacement reactions of l-substituent in cysteine, serine, and related analogs (8,9,18,19,25). As a rule, these lyases, e.g., cysteine lyase (EC 4.4.1.10), serine sulfhydrase, and the identical or closely similar cystathionine j3-synthase (EC 4.2.1.22), use aliphatic thiols as cosubstrates (replacing agents). It seemed of particular interest to extend our studies to (CN)Ala synthase, a jB-lyase utilizing cyanide as the preferred cosubstrate.Abbreviations: Pyridoxal-P, pyridoxal-5'-phosphate; (CN)Ala, fl-cyano-L-alanine; (Cl)Ala, B-chloroalanine; (CNS)Ala, fl-thiocyano-i>alanine; HS-EtOH, 2-mercaptoethanol; r.s.r., relative specific radioactivity. 1617 In this paper, we present an improved purification procedure for (CN)Ala synthase, and report on some general and catalytic properties of the enzyme.
MATERIALS AND METHODSReagents and Instruments. All chemicals were preparations of highest purity available, namely: DEAE-Sephadex A-50 and Sephadex G-100 (Pharmacia, Uppsala); hydroxylapatite from Bio-Rad; pyridoxal-P (Reanal, Budapest); dansyl chloride (Merck); reference dansylamino acids (from "Serva"); 3H20 (61 mCi/ml), made in USSR. Aluminum hydroxide C.) was prepared according to Willstatter and Kraut (27). We thank Dr. Drell (Calbiochem) for the gift of a sample of ,B-cyanoalanine.Reagents and apparatus for analytical gel electrophoresis were supplied by Reanal; Ampholine buffers and equipment for isoelectrofocusing, by LKB Producter (Stockholm).Chromatographic and electrophoretic separations were performed on papers FN-4 and FN-16 ("Filtrak", German Democratic Republic). All buffer solutions used were Tris HC1 of pH 8.8, in the concentrations indicated.Enzyme Activity Assays. (CN)Ala synthase activity assays were based on the rates of formation of end-products-H2S, (CN
