Evidence for overlapping active sites in a multifunctional enzyme: immunochemical and chemical modification studies on C1-tetrahydrofolate synthase from Saccharomyces cerevisiae

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Abstract
The relationship of the active sites which catalyze the 3 reactions in the trifunctional enzyme C1-tetrahydrofolate synthetase (C1-THF synthase) from S. cerevisiae was examined with immunochemical and chemical modification techniques. Immunotitration of the enzyme with a polyclonal antiserum resulted in identical inhibition curves for the dehydrogenase and cyclohydrolase activities which were distinctly different from the inhibition curve for the synthease activity. During chemical modification with diethyl pyrocarbonate (DEPC), the 3 activities were inactivated at significantly different rates, indicating that at least 3 distinct essential residues are involved in the reaction with DEPC. The pH dependence of the reaction with DEPC was consistent with the modification of histidyl residues. Treatment of C1-THF synthase with N-ethylmaleimide (NEM) resulted in significant inactivation of only the dehydrogenase and cyclohydrolase activities, with the cyclohydrolase at least an order of magnitude more sensitive than the dehydrogenase. Inactivation of cyclohydrolase was biphasic at NEM concentrations above 0.1 mM, suggesting 2 essential cysteinyl residues were being modified. NADP+, a dehydrogenase substrate, protecting both dehydrogenase and cyclohydrolase activities, but not synthetase activity, against inactivation by either reagent. Synthetase substrates had no protective ability. Pteroylpolyglutamates and p-aminobenzoic acid polyglutamates exhibited some protection of all 3 activities. The p-aminobenzoic acid polyglutamate series showed progressive protection with increasing chain length. These results are consistent with an overlapping site for the dehydrogenase and cyclohydrolase reactions, independent from the synthetase active site. Possible active-site configurations and the role of the polyglutamate tail in substrate binding are discussed.