A highly reactive beta-galactosidase (Escherichia coli) resulting from a substitution of an aspartic acid for Gly-794.
The beta-galactosidases of several mutagenized strains of Escherichia coli K12 which grew on lactobionate were found to be heat labile. Sequence analysis of the lacZ gene (ligated into Bluescript) of one of these strains (E. coli REH4) showed that the only change in the amino acid sequence was a substitution of an Asp for Gly-794. This change caused a dramatic increase of the activity when lactose was the substrate. The kcat of the purified enzyme from E. coli REH4 (G794D-beta-galactosidase) with lactose as the substrate was five to six times as large as the kcat of the normal enzyme with lactose. Purified G794D-beta-galactosidase was, however, less stable to heat and also to chymotrypsin (which cleaves next to Trp-585) than was normal beta-galactosidase. G794D-beta-Galactosidase bound substrates and substrate analog inhibitors less well than did normal beta-galactosidase while planar transition state analog inhibitors were more strongly bound. The ability to bind 2-amino-D-galactose (a positively charged transition state analog inhibitor) was either unaltered or was decreased somewhat. The data showed that the alteration in structure caused an increase in the value of k2 (the rate constant for the step in which the glycosidic bond is cleaved) with each substrate tested (the increase was at least 25-fold when lactose was the substrate) while k3 was decreased about 4-fold (k3 is the rate constant for the common hydrolysis step with each substrate). Since k2 is rate determining when lactose is the substrate of the normal enzyme, the increase in k2 resulted in a large increase in rate despite the fact that the value of k3 decreased. Large rate increases were not found with the other two substrates because the k2 values were not increased by large factors and because the decrease in the value of k3 negated the effects of the increased k2 values. The destabilization of the substrate binding coupled with a stabilization of the binding of a planar transition state is a possible cause of the significant increase in the value of k2 and of the enhanced activity with lactose.
Martinez-Bilbao
M
M
Division of Biochemistry, Faculty of Science, University of Calgary, Alberta, Canada.
Holdsworth
R E
RE
Edwards
L A
LA
Huber
R E
RE
D001224:Aspartic Acid / Q000235:genetics; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D005998:Glycine / Q000235:genetics; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D016296:Mutagenesis; D010957:Plasmids; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D001224:Aspartic Acid; D001616:beta-Galactosidase; D007785:Lactose; D005998:Glycine