HUMAN BETA -CELL GLUCOKINASE : DUAL ROLE OF SER-151 IN CATALYSIS AND HEXOSE AFFINITY

摘要: Glucokinase is distinguished from yeast hexokinase and low Km mammalian hexokinases by its affinity for glucose cooperative behavior, even though binding residues catalytic are highly conserved in all of these forms hexokinase. The roles Ser-151 Asn-166 as determinants hexose behavior human glucokinase have been evaluated site-directed mutagenesis, expression purification the wild-type mutant enzymes, steady-state kinetic analysis. Mutation to arginine increased apparent both ATP a factor 3. cysteine, alanine, or glycine lowered factors 2-, 26-, 40-fold, respectively, decreased Vmax, abolished cooperativity glucose, also mannose fructose. mutants had values similar those hexokinase, I, recombinantly expressed COOH-terminal half I. However, Ki competitive inhibitors, N-acetylglucosamine glucose-6-P, were unchanged, suggesting that not important inhibitor binding. about 5-fold enzyme's ATPase activity, which indicates it essential hydrolysis. substrate-induced change intrinsic fluorescence S151A occurred at much lower concentration than enzyme. results implicate dual role determinant catalysis, exclusive glucose-induced conformational change, suggest dependent on interaction with other regions protein.