Site-directed mutagenesis of glutamate-166 in beta-lactamase leads to a branched path mechanism.
Glutamate-166 of the Bacillus licheniformis beta-lactamase was specifically mutated to aspartate and cysteine in order to probe the function of this residue in catalysis. In both cases, a large decrease in activity (kcat/Km was 3.5 x 10(-5) smaller for E166C and 1 x 10(-3) smaller for E166D than for the wild-type) was observed, although the kinetics for the two mutants were very different. The pH-rate profiles for E166D and E166C reflected the ionization characteristics of the new residue at site 166. This result indicates that the ionization of Glu-166 is responsible for the acidic limb of the kcat/Km-pH profiles, and suggests that the function of Glu-166 is that of a general base catalyst. The kinetics of the E166C mutant were investigated in detail. An initial burst was observed, whose amplitude was stoichiometric with the enzyme concentration, suggesting rate-limiting deacylation of the acyl-enzyme intermediate. However, further study revealed that in the presence of 0.5 M sodium sulfate, which stabilizes the native conformational state, the magnitude of the burst corresponded to 2 equiv of enzyme. This observation, in conjunction with the limited effect of the mutation on Km, indicated that the mutation resulted in a change in the kinetic mechanism from the linear, acyl-enzyme pathway to one with a branch leading to an inactive form of the acyl-enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Escobar
W A
WA
Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064.
Tan
A K
AK
Lewis
E R
ER
Fink
A L
AL
D000215:Acylation; D001407:Bacillus / Q000201:enzymology; D001483:Base Sequence; D002384:Catalysis; D002942:Circular Dichroism; D005971:Glutamates / Q000737:chemistry / Q000235:genetics*; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation / Q000187:drug effects; D013329:Structure-Activity Relationship; D013431:Sulfates / Q000494:pharmacology; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D005971:Glutamates; D013431:Sulfates; C012036:sodium sulfate; D018698:Glutamic Acid; D001618:beta-Lactamases; D008239:Lysine