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 rev ersible cleavage of fructose 1,6-bisphosphate into dihydroxyacetone phospha te and glyceraldehyde 3-phosphate. The Class I aldolases use Schiff base fo rmation 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 s equence alignments of the Class II family of enzymes with examination of th e crystal structure of the enzyme to highlight potentially important aspart ate 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 intermed iate carbanion to investigate partial reactions. The results showed only mi nor changes in the kinetic parameters for the Asp144, Asp288, Asp290 and As p329 mutants, suggesting that these residues play only minor or indirect ro les in catalysis. By contrast, mutation of Asp109 or Asn286 caused 3000-fol d and 8000-fold decreases in the k(cat) of the reaction, respectively. Coup led with the kinetics measured for the partial reactions the results clearl y demonstrate a role for Asn286 in catalysis and in binding the ketonic end of the substrate. Fourier transform infra-red spectroscopy of the wild-typ e and mutant enzymes has further delineated the role of Asp109 as being cri tically involved in the polarisation of the carbonyl group of glyceraldehyd e 3-phosphate. (C) 1999 Academic Press.