Modulation of cyanoalanine synthase and O-acetylserine (thiol) lyases A and B activity by beta-substituted alanyl and anion inhibitors.
The reaction mechanisms of three enzymes belonging to a single gene family are compared: a cyanoalanine synthase and two isoforms of O-acetylserine (thiol) lyase (O-ASTL) isolated from spinach (Spinacea oleracea L. cv. Medina). O-ASTL represents a major regulatory point in the S-assimilatory pathway, and the related cyanoalanine synthase, which is specific to the mitochondrial compartment, has evolved an independent function of cyanide detoxification. All three enzymes catalysed both the cysteine synthesis and cyanoalanine synthesis reactions although with different efficiencies, and which may be explained by a single amino acid substitution in the substrate-binding pocket of the enzyme. Substituted alanine and nucleophillic inhibitors caused predominantly non-competitive inhibition, indicating binding to both E- and F-forms of the enzyme in a bi-bi ping-pong kinetic model. Michaelis-Menten kinetics were observed when the alanyl substrate was varied in the presence and absence of inhibitors. The use of alanyl inhibitors has shown that the alanyl half-cycle of both the cysteine synthesis and cyanoalanine synthesis reactions of cyanoalanine synthase and O-acetylserine (thiol) lyases are similar. This is in contrast to the results observed with nucleophillic inhibitors, which have shown that the mechanisms of anion binding and processing differ between cyanoalanine synthase and O-ASTLs.
Warrilow
Andrew G S
AG
IACR-Rothamsted, Agriculture and Environment Division, Harpenden, Hertfordshire AL5 2JQ, UK.
Hawkesford
Malcolm J
MJ
D000409:Alanine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D000494:Allosteric Regulation; D000495:Allosteric Site; D000838:Anions / Q000494:pharmacology; D019757:Carbon-Oxygen Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000096:biosynthesis; D003547:Cysteine Synthase; D019281:Dimerization; D006710:Homocysteine / Q000494:pharmacology; D006860:Hydrogen Bonding; D007425:Intracellular Membranes / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008190:Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D008954:Models, Biological; D009097:Multienzyme Complexes; D011190:Potassium Cyanide / Q000494:pharmacology; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine / Q000031:analogs & derivatives* / Q000494:pharmacology; D018724:Spinacia oleracea / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics
D000838:Anions; D007527:Isoenzymes; D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins; D006710:Homocysteine; D012694:Serine; C004631:3-cyanoalanine; D003547:Cysteine Synthase; C103381:MET17 protein, S cerevisiae; C018725:O-acetylhomoserine (thiol)-lyase; D008190:Lyases; D019757:Carbon-Oxygen Lyases; C019955:beta-cyanoalanine synthase; C043943:O-acetylserine; D003545:Cysteine; D011190:Potassium Cyanide; D000409:Alanine