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, Kosaku Uyeda

Research output: Contribution to journalArticle

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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 + 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.

Original languageEnglish (US)
Pages (from-to)2166-2175
Number of pages10
JournalJournal of Biological Chemistry
Volume274
Issue number4
DOIs
StatePublished - Jan 22 1999

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Phosphofructokinase-2
Histidine
Mutation
Catalysts
Enzymes
Rats
Phosphotransferases
Phosphates
Water
Enzyme activity
Phosphoric Monoester Hydrolases
Alanine
Hydroxyl Radical
Liver
Arginine
Hydrolysis
Testis
Degradation
Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Reaction mechanism of fructose-2,6-bisphosphatase : A mutation of nucleophilic catalyst, histidine 256, induces an alteration in the reaction pathway. / Mizuguchi, Hiroyuki; Cook, Paul F.; Tai, Chia Hui; Hasemann, Charles A.; Uyeda, Kosaku.

In: Journal of Biological Chemistry, Vol. 274, No. 4, 22.01.1999, p. 2166-2175.

Research output: Contribution to journalArticle

Mizuguchi, Hiroyuki ; Cook, Paul F. ; Tai, Chia Hui ; Hasemann, Charles A. ; Uyeda, Kosaku. / Reaction mechanism of fructose-2,6-bisphosphatase : A mutation of nucleophilic catalyst, histidine 256, induces an alteration in the reaction pathway. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 4. pp. 2166-2175.
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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 + 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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T2 - A mutation of nucleophilic catalyst, histidine 256, induces an alteration in the reaction pathway

AU - Mizuguchi, Hiroyuki

AU - Cook, Paul F.

AU - Tai, Chia Hui

AU - Hasemann, Charles A.

AU - Uyeda, Kosaku

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N2 - 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.

AB - 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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