IDENTIFICATION OF ARGININE-331 AS AN IMPORTANT ACTIVE-SITE RESIDUE INTHE CLASS-II FRUCTOSE-1,6-BISPHOSPHATE ALDOLASE OF ESCHERICHIA-COLI

Treatment of the Class II fructose-1,6-bisphosphate aldolase of Escher ichia coli with the arginine-specific alpha-dicarbonyl reagents, butan edione or phenylglyoxal, results in inactivation of the enzyme. The en zyme is protected from inactivation by the substrate, fructose 1,6-bis phosphate, or by inorganic phosphate. Modification with [7-C-14] pheny lglyoxal in the absence of substrate demonstrates that enzyme activity is abolished by the incorporation of approximately 2 moles of reagent per mole of enzyme. Sequence alignment of the eight known Class II FB P-aldolases shows that only one arginine residue is conserved in all t he known sequences. This residue, Arg-331, was mutated to either alani ne or glutamic acid. The mutant enzymes were much less susceptible to inactivation by phenylglyoxal. Measurement of the steady-state kinetic parameters revealed that mutation of Arg-331 dramatically increased t he K-m for fructose 1,6-bisphosphate. Comparatively small differences in the inhibitor constant K-i for dihydroxyacetone phosphate or its an alogue, 2-phosphoglycolate, were found between the wild-type and mutan t enzymes. In contrast, the mutation caused large changes in the kinet ic parameters when glyceraldehyde 3-phosphate was used as an inhibitor . Kinetic analysis of the oxidation of the carbanionic aldolase-substr ate intermediate of the reaction by hexacyanoferrate (III) revealed th at the K-m for dihydroxyacetone phosphate was again unaffected, wherea s that for fructose 1,6-bisphosphate was dramatically increased. Taken together, these results show that Arg-331 is critically involved in t he binding of fructose bisphosphate by the enzyme and demonstrate that it interacts with the C-6 phosphate group of the substrate.