A MULTISTEP PROCESS IS RESPONSIBLE FOR PRODUCT-INDUCED INACTIVATION OF GLUCOSE-FRUCTOSE OXIDOREDUCTASE FROM ZYMOMONAS-MOBILIS

Glucose-fructose oxidoreductase from the bacterium Zymomonas mobilis c atalyzes a transhydrogenation reaction in which D-fructose reduction t o D-sorbitol is coupled to the oxidation of D-glucose or other aldoses to the corresponding aldonolactones. Tightly protein-bound NADP(H) se rves as the cofactor. We found that the interaction of glucose-fructos e oxidoreductase with its aldonolactone product triggered a sequential process that affects the protein structure conformationally and chemi cally and, ultimately, results in an irreversible loss of enzyme activ ity. (1) Probably as a mechanistic requirement during the catalytic cy cle? conformational realignments in glucose-fructose oxidoreductase ar e induced by binding of the lactone and are manifested by a 1.7-fold i ncreased accessibility to iodide quenching of the fluorescence of the active-site-bound NADPH, the exposure of one reactive cysteine (likely Cys127) and strongly red-shifted tryptophan fluorescence. (2) As a fa st subsequent reaction in vitro, the cysteine residue is deactivated, thus leading to a local: structural destabilization of glucose-fructos e oxidoreductase that, without affecting enzyme activity, leads to two fold tryptophan fluorescence as well as the exposure of three further cysteine residues, (3) The completed deactivation of these cysteines i s accompanied by a twofold increase in hydrophobic surface and thus ag gregation of the glucose-fructose oxidoreductase tetramer. Aggregation , but not release of the tightly bound NADP(H), ultimately leads to th e loss of activity and completes the inactivation of glucose-fructose oxidoreductase. Apparently small conformational changes at the NADP(H) -binding site of glucose-fructose oxidoreductase trigger high-order pr otein associations and seem to be thus responsible for an incorrect ol igomerization of the enzyme.