SHARED ACTIVE-SITES OF FRUCTOSE-1,6-BISPHOSPHATASE - ARGININE-243 MEDIATES SUBSTRATE-BINDING AND FRUCTOSE 2,6-BISPHOSPHATE INHIBITION

The active site of pig kidney fructose-1,6-bisphosphatase (EC 3.1.3.11 ) is shared between subunits, Arg-243 of one chain interacting with fr uctose-1,6-bisphosphate or fructose-2,6-bisphosphate in the active sit e of an adjacent chain. In this study, Arg-243 was replaced by alanine using techniques of site-specific mutagenesis and the cloned pig kidn ey enzyme expressed in Escherichia coli. Compared with wild-type enzym e, kinetic parameters of the altered enzyme characterizing catalytic e fficiency, magnesium binding, and inhibition by AMP differed but by le ss than an order of magnitude; affinity for substrate fructose 1,6-bis phosphate was 10-fold poorer and affinity for inhibitor fructose 2,6-b isphosphate was 1000-fold poorer. Molecular dynamics simulations were undertaken to determine possible alterations in active sites of the en zyme due to replacement of Arg-243 by Ala and suggested that in the mu tant enzyme loss of one cationic group leads to reorganization of the active site especially involving lysine residues 269 and 274. The diff erences in properties of the mutant enzyme indicate the key importance of Arg-243 in the function of fructose-1,6-bisphosphatase and confirm on a functional basis the shared active site in this important metabo lic enzyme.