Isotopic Hydrogen Exchange in Reactions Catalysed by Cysteine Lyase and Serine Sulphhydrase

Tolosa, E.A.; Maslova, R.N.; Goryachenkova, E.V.; Willhardt, Ingo; Braunstein, A. E.

doi:10.1111/j.1432-1033.1975.tb04083.x

Cited by 14 publications

(4 citation statements)

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“…3) are fairly similar to those recently reported by Tolosa et al (25) for serine sulfhydrase and cysteine lyase. With these two fl-replacing lyases, in marked contrast to the a,fl-eliminating or plurifunctional ones (e.g., tryptophanase, y-cystathionase), it was found that isotopic H exchange (1) requires the presence of adequate cosubstrate, (2) mostly is not catalyzed in substrate analogs that do not undergo the complete reaction, (3) is limited to the a-H atom, (4) its rate is of the same order as that of the From these and other properties of the selectively f-replacing lyases, it was inferred that they do not comply to…”

Section: Discussionsupporting

confidence: 79%

“…Evidence reported earlier (8,9,25) demonstrated that some pyridoxal-P-dependent enzymes playing important roles in the metabolism of hydroxy and sulfur amino acids, such as cysteine lyase, serine sulfhydrase, or cystathionine f-synthase, belong to a special subgroup of lyases which catalyze exclu- (Table 2).…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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Beta-cyanoalanine synthase: purification and characterization.

Akopyan¹,

Braunstein²,

Goryachenkova³

1975

Proc. Natl. Acad. Sci. U.S.A.

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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

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Section: Discussionsupporting

confidence: 79%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Beta-cyanoalanine synthase: purification and characterization.

Akopyan¹,

Braunstein²,

Goryachenkova³

1975

Proc. Natl. Acad. Sci. U.S.A.

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“…However, the mechanism of free ␤-CNAla synthesis is quite different from that of the ␤-CNAla residue formation reported here: free ␤-CNAla is synthesized by ␤-re-placement of the mercapto group in cysteine by cyanide (25), whereas the ␤-CNAla residue is formed by ␣,␤-elimination of the imidate as shown in Scheme 2.…”

Section: Determination Of the Structure Causing The 73-da Increase-tomentioning

confidence: 86%

Novel Catalytic Mechanism of Nucleophilic Substitution by Asparagine Residue Involving Cyanoalanine Intermediate Revealed by Mass Spectrometric Monitoring of an Enzyme Reaction

Ichiyama¹,

Kurihara²,

Li³

et al. 2000

Journal of Biological Chemistry

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. This enzyme has also been utilized as an industrial biocatalyst for the synthesis of chiral compounds based on its stereospecificity (2). We have studied intensely the reaction mechanism and structure of L-DEX YL, and revealed that the reaction begins with nucleophilic attack of Asp 10 on the ␣-carbon atom of L-2-haloalkanoates, causing the release of a halide ion and the formation of an ester intermediate (Scheme 1, I), which is subsequently hydrolyzed (3, 4). In the x-ray structure, there is a water molecule close to Asp 10 , Ser 175 , Asn 177 , and Asp 180 , the latter three of which are supposed to enhance the nucleophilicity of the water molecule for hydrolysis of the ester intermediate (5) (Scheme 1). These enzymological studies of L-DEX YL have provided critical information on the structures and functions of various hydrolases including P-type ATPases, which have a significant sequence similarity with L-DEX YL and are proposed to have the haloacid dehalogenase fold (6 -9). Recent studies on the crystal structure of Ca 2ϩ -ATPase from sarcoplasmic reticulum revealed that this ATPase has a high threedimensional structural similarity to that of L-DEX YL and many essential amino acid residues are conserved between these two proteins (10).In the course of the studies of L-DEX YL, we surprisingly found that replacement of the catalytic residue, Asp 10 , by Asn did not completely inactivate the enzyme, whereas replacement by Ala, Gly, Ser, or Glu resulted in total inactivation (11). One possible explanation is that the activity of the D10N preparation is attributed to the post-translational production of the wild-type enzyme by deamidation of Asn 10 : deamidation of Asn to produce Asp has been shown to occur in a variety of proteins (12, 13). However, it is also possible that the D10N enzyme itself catalyzes the hydrolysis of the substrate.In the present study, we analyzed the mechanism of dehalogenation catalyzed by the L-DEX YL D10N preparation by ion-spray mass spectrometry (MS), and found that the D10N enzyme itself, in addition to the wild-type enzyme produced by deamidation of the D10N enzyme, catalyzes dehalogenation. Asn 10 was shown to undergo a unique structural change in the course of the catalysis: it is converted into a ␤-cyanoalanine (␤-CNAla) residue via an imidate, and then regenerated in the catalytic cycle. This is the first report of the formation of a ␤-CNAla residue as an enzyme reaction intermediate. Also, the results demonstrate the validity of mass spectrometry in enzyme dynamics.

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