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
Mh. Yuen et al., 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, J BIOL CHEM, 274(4), 1999, pp. 2176-2184
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.