Reaction mechanism of fructose-2,6-bisphosphatase - A mutation of nucleophilic catalyst, histidine 256, induces an alteration in the reaction pathway

A bifunctional enzyme, fructose-6-phosphate,2-kinase/fructose 2,6-bisphosph atase (Fru-6-P,2-kinase/Fru-2,6-Pase), catalyzes synthesis and degradation of fructose a,6-bisphosphate (Fru-2,6-P-2). Previously, the rat liver Fru-2 ,6-Pase reaction (Fru-2,6-P-2 --> Fru-6-P + P-i) has been shown to proceed via a phosphoenzyme intermediate with His(258) phosphorylated, and mutation of the histidine to alanine resulted in complete loss of activity (Tauler, A., Lin, K., and Pilkis, S. J. (1990) J. Biol. Chem, 265, 15617-15622). In the present study, it is shown that mutation of the corresponding histidin e (His(256)) Of the rat testis enzyme decreases activity by less than a fac tor of 10 with a k(cat) of 17% compared with the wild type enzyme. Mutation of His(390) (in close proximity to His(256)) to Ala results in a k(cat) of 12.5% compared with the wild type enzyme. Attempts to detect a phosphohist idine intermediate with the H256A mutant enzyme were unsuccessful, but the phosphoenzyme is detected in the wild type, H390A, R255A, R305S, and E325A mutant enzymes. Data demonstrate that the mutation of His(256) induces a ch ange in the phosphatase hydrolytic reaction mechanism. Elimination of the n ucleophilic catalyst, H256A, results in a change in mechanism. In the H256A mutant enzyme, His(390) likely acts as a general base to activate water fo r direct hydrolysis of the a-phosphate of Fru-2,6-P-2. Mutation of Arg(255) and Arg(305) suggests that the arginines probably have a role in neutraliz ing excess charge on the a-phosphate and polarizing the phosphoryl for subs equent transfer to either His(256) or water. The role of Glu(325) is less c ertain, but it may serve as a general acid, protonating the leaving 2-hydro xyl of Fru-2,6-P-2.