2bif
6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-2,6-BISPHOSPHATASE H256A MUTANT WITH F6P IN PHOSPHATASE ACTIVE SITE
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OverviewOverview
Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase (Fru-6-P, 2-kinase/Fru-2,6-Pase) is a bifunctional enzyme, catalyzing the, interconversion of beta-D-fructose- 6-phosphate (Fru-6-P) and, fructose-2,6-bisphosphate (Fru-2,6-P2) at distinct active sites. A mutant, rat testis isozyme with an alanine replacement for the catalytic histidine, (H256A) in the Fru-2,6-Pase domain retains 17% of the wild type activity, (Mizuguchi, H., Cook, P. F., Tai, C-H., Hasemann, C. A., and Uyeda, K., (1998) J. Biol. Chem. 274, 2166-2175). We have solved the crystal, structure of H256A to a resolution of 2. 4 A by molecular replacement., Clear electron density for Fru-6-P is found at the Fru-2,6-Pase active, site, revealing the important interactions in substrate/product binding. A, superposition of the H256A structure with the RT2K-Wo structure reveals no, significant reorganization of the active site resulting from the binding, of Fru-6-P or the H256A mutation. Using this superposition, we have built, a view of the Fru-2,6-P2-bound enzyme and identify the residues, responsible for catalysis. This analysis yields distinct catalytic, mechanisms for the wild type and mutant proteins. The wild type mechanism, would lead to an inefficient transfer of a proton to the leaving group, Fru-6-P, which is consistent with a view of this event being, rate-limiting, explaining the extremely slow turnover (0. 032 s-1) of the, Fru-2,6-Pase in all Fru-6-P,2-kinase/Fru-2,6-Pase isozymes.
About this StructureAbout this Structure
2BIF is a Single protein structure of sequence from Rattus norvegicus with BOG, F6P, MG, PO4, ANP and SIN as ligands. Full crystallographic information is available from OCA.
ReferenceReference
Crystal structure of the H256A mutant of rat testis fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase. Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities., Yuen MH, Mizuguchi H, Lee YH, Cook PF, Uyeda K, Hasemann CA, J Biol Chem. 1999 Jan 22;274(4):2176-84. PMID:9890980
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