Identification of arginine 331 as an important active site residue in the class II fructose-1,6-bisphosphate aldolase of Escherichia coli.

Treatment of the Class II fructose-1,6-bisphosphate aldolase of Escherichia coli with the arginine-specific alpha-dicarbonyl reagents, butanedione or phenylglyoxal, results in inactivation of the enzyme. The enzyme is protected from inactivation by the substrate, fructose 1,6-bisphosphate, or by inorganic phosphate. Modification with [7-14C] phenylglyoxal 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 FBP-aldolases shows that only one arginine residue is conserved in all the known sequences. This residue, Arg-331, was mutated to either alanine 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 the K(m) for fructose 1,6-bisphosphate. Comparatively small differences in the inhibitor constant Ki for dihydroxyacetone phosphate or its analogue, 2-phosphoglycolate, were found between the wild-type and mutant enzymes. In contrast, the mutation caused large changes in the kinetic parameters when glyceraldehyde 3-phosphate was used as an inhibitor. Kinetic analysis of the oxidation of the carbanionic aldolase-substrate intermediate of the reaction by hexacyanoferrate (III) revealed that the K(m) for dihydroxyacetone phosphate was again unaffected, whereas that for fructose 1,6-bisphosphate was dramatically increased. Taken together, these results show that Arg-331 is critically involved in the binding of fructose bisphosphate by the enzyme and demonstrate that it interacts with the C-6 phosphate group of the substrate.