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.
Plater
A R
AR
School of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2 9JT, England.
Zgiby
S M
SM
Thomson
G J
GJ
Qamar
S
S
Wharton
C W
CW
Berry
A
A
D004926:Escherichia coli / Q000201:enzymology*; D005295:Ferrocyanides / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D017550:Spectroscopy, Fourier Transform Infrared
D005295:Ferrocyanides; C116383:ferrocyn; D005634:Fructose-Bisphosphate Aldolase