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J Biol Chem, Vol. 274, Issue 4, 2166-2175, January 22, 1999

Reaction Mechanism of Fructose-2,6-bisphosphatase
A MUTATION OF NUCLEOPHILIC CATALYST, HISTIDINE 256, INDUCES AN ALTERATION IN THE REACTION PATHWAY

Hiroyuki Mizuguchi, Paul F. Cook, Chia-Hui Tai, Charles A. Hasemann^§, and Kosaku Uyeda

From Research Service, Dallas Veterans Affairs Medical Center, Dallas, Texas 75216, the ^§ Departments of Biochemistry and Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75216, and the  Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019

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 + P_i) 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 k_cat of 17% compared with the wild type enzyme. Mutation of His³⁹⁰ (in close proximity to His²⁵⁶) to Ala results in a k_cat 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.

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