By using site-directed mutagenesis, the conserved Lys-67 residue situated three positions after the active-site Ser of a class C beta-lactamase was replaced by Arg or Gln. The Lys-67-Gln protein was nearly inactive. Although severely impaired, the Lys-67-Arg mutant exhibited an appreciable activity above pH 7.5 and, for some poor substrates of the wild-type enzyme, the kcat. values were even increased. The properties of the Lys-67-Arg mutant were studied by both steady-state and transient-state kinetic methods with a variety of compounds representing distinct classes of available substrates. With beta-lactam substrates, the kcat./Km values reflecting the efficiency of the acylation step (k+2/K) were decreased 25-100-fold. When the individual values could be measured, k+2 was not significantly altered, but K was found to be strongly increased, a result most likely explained by a corresponding increase in the k+1/k-1 ratio. These results, combined with the much stronger impairment of the Lys-67-Gln mutant, can be interpreted by attributing an electrostatic role to the positive ammonium group of the Lys-67 side chain.
Research Article| August 15 1994
The role of lysine-67 in a class C β-lactamase is mainly electrostatic
Biochem J (1994) 302 (1): 1–4.
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D Monnaie, A Dubus, J M Frère; The role of lysine-67 in a class C β-lactamase is mainly electrostatic. Biochem J 15 August 1994; 302 (1): 1–4. doi: https://doi.org/10.1042/bj3020001
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