Substitutions for Glu-537 of beta-galactosidase from Escherichia coli cause large decreases in catalytic activity.
Glu-537 of beta-galactosidase (EC 3.2.1.23) was replaced by Asp, Gln and Val using synthetic oligonucleotides. The kcat values of the purified enzyme mixtures were reduced by about 100-fold for the Asp mutant, 30,000-60,000-fold for the Val mutant and 160,000-300,000-fold for the Gln mutant. The greatest differences in properties from the wild-type enzyme were found for the Asp-substituted enzyme: the Km values increased (from 0.12 to 0.42 mM for o-nitrophenyl beta-D-galactopyranoside), and from 0.04 to 0.37 mM for p-nitrophenyl beta-D-galactopyranoside), the Ki value for isopropyl beta-D-galactopyranoside increased (from 0.11 to 0.30 mM), the stability to heat decreased and methanol did not act as an acceptor. The enzymes with the other two substitutions had properties similar to those of the wild-type. For all three substituted enzymes, the inhibitory effects of the transition-state analogues (2-deoxy-2-amino-D-galactose and L-ribose) and the Mg2+ effects were similar to those of the normal enzyme. As all of the properties (except the kcat values) of the Gln- and Val-substituted enzyme preparations were similar to those of the wild-type enzyme, the activities in those preparations were probably due to the presence of a few wild-type enzyme molecules (formed from misreads) among the substituted enzymes. The enzymes with Gln and Val substitutions appear to be totally inactive. The results obtained support a recent suggestion that Glu-537 is an important catalytic residue of beta-galactosidase.
Yuan
J
J
Division of Biochemistry, Faculty of Science, University of Calgary, Alberta, Canada.
Martinez-Bilbao
M
M
Huber
R E
RE
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D003062:Codon; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D001616:beta-Galactosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D003062:Codon; D017931:DNA Primers; D005971:Glutamates; D011994:Recombinant Proteins; D018698:Glutamic Acid; D001616:beta-Galactosidase