Evidence for a sulfhydryl group at the active site of serine transhydroxymethylase.

Iodoacetate reacts rapidly with one sulfhydryl group/subunit on aposerine transhydroxymethylase. The carboxymethylated apoenzyme does not recombine with pyridoxal 5'-phosphate. Under conditions used in the apoenzyme studies, the holoenzyme does not react to an appreciable extent with iodoacetate. The reaction of the apoenzyme with iodoacetate shows pseudo-first order kinetics with a half-life of about 3 min at 0 degrees C and pH 7.0. A pattern of saturation kinetics was found when increasing concentrations of iodoacetate were used. The half-maximum rate of inactivation occurred at 1.5 mM iodoacetate. Phosphate was observed to inhibit competitively the inactivation by iodoacetate with a Ki value of 1.8 mM. No inactivation of aposerine transhydroxymethylase was found when iodoacetamide was used in place of iodoacetate. These experiments suggest that removal of the pyridoxal 5'-phosphate from serine transhydroxymethylase exposes a reactive sulfhydryl group with a nearby cationic center which binds the carboxyl group of iodoacetate. The reactive sulfhydryl group was labeled with [14C]iodoacetate. From a chymotryptic digest, a 14C-containing peptide was isolated and determined to be: His-Pro-Lys-Leu-Ile-Ile-Ala-Gly-Thr-Ser-Cys(Cm)-Tyr.