Inactivation of Soluble Bovine Milk Galactosyltransferase (Lactose Synthetase) by Sulfhydryl Reagents and Trypsin

Abstract

Abstract Galactosyltransferase exhibited a time-dependent loss of activity when incubated with sulfhydryl reagents p-chloromercuribenzoate and N-ethylmaleimide. Though incubations were continued for periods 20-fold greater than the half-time of inactivation, neither reagent eliminated all catalytic activity, and generally 5 to 10% of the initial activity remained. p-Chloromercuribenzoate inactivated the enzme more rapidly then N-ethylmaleimide, but the inhibition by p-chloromercuribenzoate was reversed by mercaptoethanol. Substrates and substrate analogs protected against inhibition by p-chloromercuribenzoate and N-ethylmaleimide. Ligands giving the most protection against inhibition were analogs of the nucleotide substrate and product, uridine diphosphate galactose and UDP, respectively, in the presence of manganous chloride. When incubated with 10 mm N-ethylmaleimide, galactosyltransferase exhibited an apparent rate constant of inactivation of 0.023 min-1; adding analogs of UDP at concentrations of 2 mm reduced this rate by 30% and the same analogs in the presence of manganous chloride reduced the rate constant of inactivation by 98%. Inhibition of galactosyltransferase by N-ethylmaleimide resulted in a dramatic decrease in Vmax whereas the apparent Km for UDP-galactose remained constant. A similar pattern of protection against inactivation of galactosyltransferase by trypsin was observed suggesting that a conformational change in the protein is responsible for the observed protection.