TY - JOUR
T1 - Crystal structure of the H256A mutant of rat testis fructose-6- phosphate,2-kinase/fructose-2,6-bisphosphatase
T2 - Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities
AU - Yuen, Mi H.
AU - Mizuguchi, Hiroyuki
AU - Lee, Yong Hwan
AU - Cook, Paul F.
AU - Uyeda, Kosaku
AU - Hasemann, Charles A.
PY - 1999/1/22
Y1 - 1999/1/22
N2 - 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 β-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 s-1) of the Fru-2,6-Pase in all Fru-6-P,2-kinase/Fru-2,6-Pase isozymes.
AB - 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 β-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 s-1) of the Fru-2,6-Pase in all Fru-6-P,2-kinase/Fru-2,6-Pase isozymes.
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U2 - 10.1074/jbc.274.4.2176
DO - 10.1074/jbc.274.4.2176
M3 - Article
C2 - 9890980
AN - SCOPUS:0033593452
SN - 0021-9258
VL - 274
SP - 2176
EP - 2184
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 4
ER -