Carboxyl terminus region modulates catalytic activity of recombinant maize aldolase.
Site-directed mutagenesis was utilized to study the functional role of the COOH-terminal region in recombinant maize aldolase. A single mutation was created in each of the last nine amino acids of the COOH terminus and characterized kinetically. Point mutations in the COOH-terminal region were found to influence both the rate of fructose 1,6-bisphosphate and fructose 1-phosphate cleavage. Catalytic efficiency, kcat/Km, was not affected by the mutations within experimental error consistent with this region of the COOH terminus modulating product release. Concentrations of the carbanion-enamine enzyme intermediate complex produced upon substrate cleavage increased with the severity of the point mutation. A condensation assay was developed to directly measure fructose 1,6-bisphosphate synthesized by aldolases in the presence of high triose phosphate concentrations. The maximal rate of aldol condensation of triose phosphates, D-glyceralehyde-3-P and dihydroxyacetone-P, was affected by the point mutations to the same extent as the maximal rate of substrate cleavage. Interpretation of the data is consistent with point mutations in the COOH terminus predominantly affecting the proton exchange with the dihydroxyacetone-P enzymatic complex at the carbanion-enamine step and that this step is probably rate-limiting in the catalytic mechanism of recombinant maize aldolase. The role of the COOH-terminal region in aldolases is thus consistent with a sequence dependent modulation of catalytic activity.
Berthiaume
L
L
Department of Biochemistry, Faculty of Medicine, Université de Sherbrooke, Québec, Canada.
Loisel
T P
TP
Sygusch
J
J
D000595:Amino Acid Sequence; D002264:Carboxylic Acids / Q000737:chemistry; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D016415:Sequence Alignment; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology*
D002264:Carboxylic Acids; D005634:Fructose-Bisphosphate Aldolase