Conserved residues in the mechanism of the E. coli Class II FBP-aldolase.

The two classes of fructose-1,6-bisphosphate aldolase both catalyse the reversible cleavage of fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The Class I aldolases use Schiff base formation as part of their catalytic mechanism, whereas the Class II enzymes are zinc-containing metalloproteins. The mechanism of the Class II enzymes is less well understood than their Class I counterparts. We have combined sequence alignments of the Class II family of enzymes with examination of the crystal structure of the enzyme to highlight potentially important aspartate and asparagine residues in the enzyme mechanism. Asp109, Asp144, Asp288, Asp290, Asp329 and Asn286 were targeted for site-directed mutagenesis and the resulting proteins purified and characterised by steady-state kinetics using either a coupled assay system to study the overall cleavage reaction or using the hexacyanoferrate (III) oxidation of the enzyme bound intermediate carbanion to investigate partial reactions. The results showed only minor changes in the kinetic parameters for the Asp144, Asp288, Asp290 and Asp329 mutants, suggesting that these residues play only minor or indirect roles in catalysis. By contrast, mutation of Asp109 or Asn286 caused 3000-fold and 8000-fold decreases in the kcat of the reaction, respectively. Coupled with the kinetics measured for the partial reactions the results clearly demonstrate a role for Asn286 in catalysis and in binding the ketonic end of the substrate. Fourier transform infra-red spectroscopy of the wild-type and mutant enzymes has further delineated the role of Asp109 as being critically involved in the polarisation of the carbonyl group of glyceraldehyde 3-phosphate.

A functional role for a flexible loop containing Glu182 in the class II fructose-1,6-bisphosphate aldolase from Escherichia coli.

Class II fructose 1,6-bisphosphate aldolases (FBP-aldolases) catalyse the zinc-dependent, reversible aldol condensation of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P) to form fructose 1,6-bisphosphate (FBP). Analysis of the structure of the enzyme from Escherichia coli in complex with a transition state analogue (phosphoglycolohydroxamate, PGH) suggested that substrate binding caused a conformational change in the beta5-alpha7 loop of the enzyme and that this caused the relocation of two glutamate residues (Glu181 and Glu182) into the proximity of the active site. Site-directed mutagenesis of these two glutamate residues (E181A and E182A) along with another active site glutamate (Glu174) was carried out and the mutant enzymes characterised using steady-state kinetics. Mutation of Glu174 (E174A) resulted in an enzyme which was severely crippled in catalysis, in agreement with its position as a zinc ligand in the enzyme's structure. The E181A mutant showed the same properties as the wild-type enzyme indicating that the residue played no major role in substrate binding or enzyme catalysis. In contrast, mutation of Glu182 (E182A) demonstrated that Glu182 is important in the catalytic cycle of the enzyme. Furthermore, the measurement of deuterium kinetic isotope effects using [1(S)-(2)H]DHAP showed that, for the wild-type enzyme, proton abstraction was not the rate determining step, whereas in the case of the E182A mutant this step had become rate limiting, providing evidence for the role of Glu182 in abstraction of the C1 proton from DHAP in the condensation direction of the reaction. Glu182 lies in a loop of polypeptide which contains four glycine residues (Gly176, Gly179, Gly180 and Gly184) and a quadruple mutant (where each glycine was converted to alanine) showed that flexibility of this loop was important for the correct functioning of the enzyme, probably to change the microenvironment of Glu182 in order to perturb its pK(a) to a value suitable for its role in proton abstraction. These results highlight the need for further studies of the dynamics of the enzyme in order to fully understand the complexities of loop closure and catalysis in this enzyme.