PURIFICATION AND KINETIC CHARACTERIZATION OF HEXOKINASE AND GLUCOSE-6-PHOSPHATE-DEHYDROGENASE FROM SCHIZOSACCHAROMYCES-POMBE

Hexokinase and D-glucose-6-phosphare dehydrogenase (G6PDH) from Schizo saccharomyces pombe have been purified 250-fold by an identical three- step. Both enzymes are dimeric with a molecular mass of 88 kDa for the kinase and 112 kDa for the dehydrogenase. Steady-stale kinetic studie s were performed on hexokinase and G6PDH, which form the glucose phosp hate branch of the oxidative pentose phosphate pathway of S. pombe (fi ssion yeast). Hexokinase promotes Mg2+-activated phosphorylation of D- glucose by the equilibrium random Bi Bi mechanism with formation of th e abortive enzyme-ADP-glucose complex. ADP inhibits the kinase competi tively versus ATP and noncompetitively versus D-glucose. The Mg2+ acti vation of hexokinase is associated with an increase in the maximal vel ocity by its interaction with the ternary complex to facilitate the tr ansfer of the phosphoryl group. G6PDH catalyzes NADP(+)-linked oxidati on of D-glucose-6-phosphate by the ordered Bi Ei mechanism with NADP() as the leading reactant. High NADP+ concentration inhibits the dehyd rogenase by forming the dead-end ternary complex. In addition, G6PDH i s also subjected to product inhibition by NADPH and noncompetitive inh ibition by A(G)TP. Thus, the oxidative pentose phosphate pathway in S. pombe may be regulated via inhibition of hexokinase by ADP in conjunc tion with inhibition of G6PDH by NADPH and ATP.