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

Abstract

Treatment of the Class 11 fructose-1.6-bisphosphate aldolase of Escherichia coli with the arginine-specific a-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-"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 I1 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,,, for fructose 1.6-bisphosphate. Comparatively small differences in the inhibitor constant K, 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 (111) revealed that the K , 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.

Structure of Tagatose-1,6-bisphosphate Aldolase

“…4, placed the C6 phosphate to interact with Arg 331 of the partner subunit and Ser 61 . These residues are conserved in the class II FBP-aldolases, and the model is consistent with the kinetic analysis of mutant enzymes in which these two residues have been altered (9,12). The C4 hydroxyl of FBP is positioned about 4 Å from Asn 35 and 3 Å from the carboxylate side chain of Asp 109 .…”

“…Receptor models for TBP aldolase and FBP aldolase were derived from the complexes with PGH with all water molecules removed. In addition, for FBPA the side chain of Glu 182 was deleted to allow access to Arg 331 , which has been shown to interact with the G3P phosphate of the substrate (12) and to be consistent with TBPA for which the corresponding residue Glu 142 is disordered and missing from the model. Suitable parameters for the Zn 2ϩ , Na ϩ , and substrate atoms were derived by reference to literature values (28) and systematic variation to reproduce the position of PGH observed in the experimentally derived crystal structures.…”

Structural Basis for Catalysis of a Tetrameric Class IIa Fructose 1,6-Bisphosphate Aldolase from Mycobacterium tuberculosis

“…The remaining H-bonds are formed with waters and the side chains of K308 and R314 which are contributed by symmetry related monomers. The importance of the interaction between R314 and the C6-phosphate of FBP and G3P has been shown through mutagenesis and modeling experiments with E. coli FBA27; 28. When the equivalent residue (R331) in E. coli is mutated to an alanine residue, a 26-fold increase in the K M value for FBP and a 29-fold decrease in k cat are observed 27.…”

Modifying the stereochemistry of an enzyme-catalyzed reaction by directed evolution

“…Despite our growing understanding of the catalytic mechanism of the class II FBP aldolase (24)(25)(26), details regarding stereochemical control remain unclear. After abstraction of the 1-pro S proton from DHAP by Glu-182 (26,27), attack of the DHAP ene-diolate intermediate on the Si-face of G3P in the FBP aldolases generates FBP (Fig.…”