[Site-directed mutagenesis of Lac Z gene in Escherichia coli and the kinetic properties of the mutated enzymes].
Glutamic acid at position of 537 of beta-D-galactosidase coded by Lac Z gene was substituted with Aspartic acid, Glutamine and Valine using synthetic oligonucleotide probes. Compared to native enzyme, the kcat values for substrate ONPG were 0.13%, 0.0006% and 0.0035% for Asp-537, Gln-537 and Val-537 mutated enzymes respectively. The Km values were of the same order of magnitude, either native or mutated enzymes. The substrate analog, IPTG was a strong inhibitor of each of the substituted enzymes, as in the case of native enzyme. The transition state analogs, 2-NH2-galactose and L-ribose were almost the same effects for the mutated enzymes as for the normal enzyme. The nucleophili, Azide, did not activate the mutated enzymes as in the case of Glu-461 substituted in beta-D-galactosidase. The effect of methanol on the mutated enzymes was less than on native enzyme. The order of the thermal stability was native enzyme > Asp-537 > Gln-537 > Val-537 enzymes. Overall, the evidence strongly supports the suggestion that Glu-537 is an essential residue of beta-D-galactosidase.
Chu
X
X
Department of Biology, Shanxi University, Taiyuan.
Bai
Y
Y
Yuan
J
J
D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D001616:beta-Galactosidase / Q000235:genetics*
D015345:Oligonucleotide Probes; D004247:DNA; D001616:beta-Galactosidase