The role of lysine-234 in .beta.-lactamase catalysis probed by site-directed mutagenesis

Abstract

Lys-234 has been postulated to participate in .beta.-lactamase catalysis by acting as an electrostatic anchor for the C3 carboxylate of penicillins [Herzberg, O., and Moult, J. (1987) Science 236, 694-701]. To test this hypothesis, site-directed mutagenesis was used to convert the Lys-234 in Bacillus licheniformis .beta.-lactamase into Glu-234 or Ala-234. The wild-type, Glu-234, and Ala-234 .beta.-lactamases have been expressed in Bacillus subtilis and purified to homogeneity. The wild-type, K234E, and K234A enzymes have virtually identical circular dichroism and fluorescence spectra, similar thermal stabilities at neutral pH, and the same susceptibilities to proteolysis, indicating the lack of significant structural perturbation caused by the mutation. At acidic and basic pH the mutant enzymes have the same native circular dichroism as the wild-type enzyme but the thermal stability is significantly different. The mutations cause perturbations of the pK values of the ionizing groups responsible for the pH dependence of the catalytic reaction in both the free enzyme and the E.S complex. As expected, conversion of Lys-234 to Ala or Glu decreased substrate binding (Km) by 1-2 orders of magnitude for several penicillin and cephalosporin substrates at neutral and higher pH. However, at low pH, Km is essentially the same for the K234E and K234A enzymes as for the wild-type enzyme. Furthermore, decreases of 2-3 orders of magnitude in kcat were also observed, indicating substantial effects on the transition-state binding, as well as on ground-state binding. Surprisingly, changing the C3 carboxylate of phenoxymethylpenicillin to a hydroxymethyl group led to little difference in kinetic properties with the K234E or k234A enzyme. The results of this investigation indicate that Lys-234 is an important active-site residue involved in both ground-state and transition-state binding.