作者: Dean R. Appling , Jesse C. Rabinowitz
DOI: 10.1021/BI00335A023
关键词:
摘要: The relationship of the active sites which catalyze three reactions in trifunctional enzyme C1-tetrahydrofolate synthase (C1-THF synthase) from Saccharomyces cerevisiae has been examined with immunochemical and chemical modification techniques. Immunotitration a polyclonal antiserum resulted identical inhibition curves for dehydrogenase cyclohydrolase activities were distinctly different curve synthetase activity. During diethyl pyrocarbonate (DEPC), inactivated at significantly rates, indicating that least distinct essential residues are involved reaction DEPC. pH dependence DEPC was consistent histidyl residues. Treatment C1-THF N-ethylmaleimide (NEM) significant inactivation only activities, an order magnitude more sensitive than dehydrogenase. Inactivation biphasic NEM concentrations above 0.1 mM, suggesting two cysteinyl being modified. NADP+, substrate, protected both but not activity, against by either reagent. Synthetase substrates had no protective ability. Pteroylpolyglutamates p-aminobenzoic acid polyglutamates exhibited some protection all activities. polyglutamate series showed progressive increasing chain length. These results overlapping site reactions, independent site. Possible active-site configurations role tail substrate binding discussed.