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

Fructose-6-phosphate,2-kinase/fructose-2 phatase (Fru-6-P,2-kinase/Fru-2,6- Pase) is a bifunctional enzyme, catalyzing the interconversion of beta-D-fr uctose-6-phosphate (Fru-6-P) and fructose-2,6-bisphosphate (Fru-2,6-P-2) at distinct active sites. A mutant rat testis isozyme with an alanine replace ment 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., Hase mann, C.A., and Uyeda, K. (1998) J. Biol Chem. 274, 2166-2175), We have sol ved the crystal structure of H256A to a resolution of 2.4 Angstrom by molec ular replacement. Clear electron density for Fru-6-P is found at the Fru-2, 6-Pase active site, revealing the important interactions in substrate/produ ct binding. A superposition of the H256A structure with the RT2K-Wo structu re 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-P-2-bound enzyme and identify the residues responsible for catalysis. This analysis yields distinct catalytic mechani sms for the wild type and mutant proteins. The wild type mechanism would le ad to an inefficient transfer of a proton to the leaving group Fru-6-P, whi ch 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.