[Resolution of beta-cyanoalanine synthase and recombination of its apoenzyme with pyridoxal-5'-phosphate and its analogs].
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.
D001051:Apoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D001059:Apoproteins / Q000302:isolation & purification*; D001665:Binding Sites; D001667:Binding, Competitive; D003545:Cysteine; D004789:Enzyme Activation; D007700:Kinetics; D008190:Lyases / Q000302:isolation & purification* / Q000378:metabolism; D009570:Nitriles; D010944:Plants / Q000201:enzymology; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000378:metabolism*
D001051:Apoenzymes; D001059:Apoproteins; D009570:Nitriles; D011732:Pyridoxal Phosphate; D008190:Lyases; C019955:beta-cyanoalanine synthase; D003545:Cysteine
Poluchenie apofermenta beta-tsianoalaninsintazy i reaktivirovanie ego piridoksal'-5'-fosfatom. iego analogami.