Abstract
A bifunctional enzyme, fructose-6-phosphate,2-kinase/fructose 2, 6-bisphosphatase (Fru-6-P,2-kinase/Fru-2,6-Pase), catalyzes synthesis and degradation of fructose 2,6-bisphosphate (Fru-2,6-P2). Previously, the rat liver Fru-2,6-Pase reaction (Fru-2,6-P2 --> Fru-6-P + Pi) has been shown to proceed via a phosphoenzyme intermediate with His258 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 histidine (His256) of the rat testis enzyme decreases activity by less than a factor of 10 with a kcat of 17% compared with the wild type enzyme. Mutation of His390 (in close proximity to His256) to Ala results in a kcat of 12.5% compared with the wild type enzyme. Attempts to detect a phosphohistidine 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 His256 induces a change in the phosphatase hydrolytic reaction mechanism. Elimination of the nucleophilic catalyst, H256A, results in a change in mechanism. In the H256A mutant enzyme, His390 likely acts as a general base to activate water for direct hydrolysis of the 2-phosphate of Fru-2,6-P2. Mutation of Arg255 and Arg305 suggests that the arginines probably have a role in neutralizing excess charge on the 2-phosphate and polarizing the phosphoryl for subsequent transfer to either His256 or water. The role of Glu325 is less certain, but it may serve as a general acid, protonating the leaving 2-hydroxyl of Fru-2,6-P2.
Highlights
A bifunctional enzyme, fructose-6-phosphate,2-kinase/ fructose 2,6-bisphosphatase (Fru-6-P,2-kinase/Fru-2,6Pase), catalyzes synthesis and degradation of fructose 2,6-bisphosphate (Fru-2,6-P2)
During the course of preparation of a mutant testis enzyme in which His[256] was substituted with Ala (which, by analogy to the liver enzyme, should have eliminated Fru-2,6-Pase activity (5)), we found that the H256A mutant enzyme retained the hydrolytic activity
In order to rule out the possibility that the preparation of the mutant enzyme was contaminated with a trace amount of the wild type enzyme, the masses of both the Wo and H256A mutant enzymes were determined using a mass spectrometer
Summary
A bifunctional enzyme, fructose-6-phosphate,2-kinase/ fructose 2,6-bisphosphatase (Fru-6-P,2-kinase/Fru-2,6Pase), catalyzes synthesis and degradation of fructose 2,6-bisphosphate (Fru-2,6-P2). The rat liver Fru-2,6-Pase reaction (Fru-2,6-P2 3 Fru-6-P ؉ Pi) 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 Consistent with the idea that a histidine residue is central to the catalytic mechanism of Fru-2,6-Pase is the observation that mutation of His[258] of the rat liver enzyme results in complete loss of the hydrolytic activity (5). The rate of formation of the phosphorylenzyme has been shown to be faster than the overall phosphatase activity, supporting a phosphoenzyme intermediate (6) These results suggested the following scheme (Scheme I) for the reaction pathway for the Fru-2,6-P2 hydrolysis catalyzed by Fru-2,6-Pase.
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