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J Biol Chem, Vol. 274, Issue 4, 2176-2184, January 22, 1999

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

Mi H. Yuen, Hiroyuki Mizuguchi^§¶, Yong-Hwan Lee, Paul F. Cook, Kosaku Uyeda^§¶, and Charles A. Hasemann^§

From the Departments of  Internal Medicine and ^§ Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235, ^¶ Research Service, Department of Veterans Affairs Medical Center, Dallas, Texas 75216, and the  Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019

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 -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 Å 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 s¹) of the Fru-2,6-Pase in all Fru-6-P,2-kinase/Fru-2,6-Pase isozymes.

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