Protein Science, Vol 5, Issue 1 154-161, Copyright © 1996 by Cold Spring Harbor Laboratory Press

ARTICLE

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

S. QAMAR, K. MARSH and A. BERRY
Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, United Kingdom Present address: Department of Biochemistry & Molecular Biology, University of Leeds, Leeds, LS2 9JT, United Kingdom.

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-(14)C] 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 K(i) 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.
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