title: Escherichia coli E-39 ADPglucose synthetase has different activation kinetics from the wild-type allosteric enzyme.
abstract: Kinetic and binding studies have shown that Lys39 of Escherichia coli ADPglucose synthetase is involved in binding of the allosteric activator. In order to study structure-function relationships at the activator binding site, this lysine residue was substituted by glutamic acid (Lys39----Glu) by site-directed mutagenesis. The resultant mutant enzyme (E-39) showed activation kinetics different from those of the wild-type enzyme. The level of activation of the E-39 enzyme by the major activators of E. coli ADPglucose synthetase, 2-phosphoglycerate, pyridoxal phosphate, and fructose-1,6-phosphatase was only approximately 2-fold compared to activation of 15- to 28-fold respectively, for the wild-type enzyme. NADPH, an activator of the wild-type enzyme, was unable to activate the mutant enzyme. In addition, the concentrations of the above activators necessary to obtain 50% of the maximal stimulation of enzyme activity (A0.5) were 5-, 9-, and 23-fold higher, respectively, than those for the wild-type enzyme. The E-39 enzyme also had a lower apparent affinity (S0.5) for the substrates ATP and MgCl2 than the wild-type enzyme and the values obtained in the presence or absence of activator were similar. The concentration of inhibitor giving 50% of enzyme activity (I0.5) was also similar for the E-39 enzyme in the presence or absence of activator. These results indicate that the E-39 mutant enzyme is not effectively activated by the major activators of the E. coli ADPglucose synthetase wild-type enzyme, and that this amino acid substitution also prevents the allosteric effect that the activator has on the wild-type enzyme kinetics, either increasing its apparent affinity for the substrates or modulating the enzyme's sensitivity to inhibition.
authors.affiliation: Department of Biochemistry, Michigan State University, East Lansing 48824.
authors.forename: A
authors.initials: A
authors.lastname: Gardiol
authors.forename: J
authors.initials: J
authors.lastname: Preiss
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D051599:Glucose-1-Phosphate Adenylyltransferase; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D009154:Mutation; D009713:Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015345:Oligonucleotide Probes
chemical_list: D015345:Oligonucleotide Probes; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase; D008239:Lysine