The role of lysine-67 in a class C beta-lactamase is mainly electrostatic.
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.
Monnaie
D
D
Centre d'ingénierie des Protéines and Laboratoire d'Enzymologie, Université de Liège, Belgium.
Dubus
A
A
Frère
J M
JM
D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D004563:Electrochemistry; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D001618:beta-Lactamases / Q000737:chemistry / Q000145:classification / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D001120:Arginine; D001618:beta-Lactamases; D008239:Lysine