[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.