THE ROLE OF LYSINE-67 IN A CLASS-C BETA-LACTAMASE IS MAINLY ELECTROSTATIC

By using site-directed mutagenesis, the conserved Lys-67 residue situa ted three positions after the active-site Sec of a class C beta-lactam ase was replaced by Arg or Gln. The Lys-67-Gln protein was nearly inac tive. Although severely impaired, the Lys-67-Arg mutant exhibited an a ppreciable activity above pH 7.5 and, for some poor substrates of the wild-type enzyme, the k(cat) values were even increased. The propertie s of the Lys-67-Arg mutant were studied by both steady-state and trans ient-state kinetic methods with a variety of compounds representing di stinct classes of available substrates. With p-lactam substrates, the k(cat)/K-m 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 s trongly increased, a result most likely explained by a corresponding i ncrease in the k(+1)/k(-1) ratio. These results, combined with the muc h stronger impairment of the Lys-67-Gln mutant, can be interpreted by attributing an electrostatic role to the positive ammonium group of th e Lys-67 side chain.