Studies on the mechanism of a class A beta-lactamase using site-directed mutagenesis and ATR-FTIR.

摘要

Thin-film ATR-FTIR was used to investigate enzyme-substrate complexes of a class A β-lactamase with various β-lactam antibiotics. This method was combined with site-directed mutagenesis of conserved amino acids in the Bacillus licheniformis enzyme to identify reaction intermediates and to determine the catalytic role of these residues.; Since β-lactamases hydrolyze β-lactams via an acyl-enzyme catalytic mechanism, ATR-FTIR was employed to monitor the stretching frequency of the substrate's β-lactam carbonyl during catalysis. The turnover reaction of poor substrates was studied with wild-type β-lactamase. The difference spectra of cefoxitin revealed the concurrent loss of substrate and steady-state presence of enzyme-bound species. The turnover of type A substrates, which are catalyzed by a branched-pathway mechanism, illustrated the presence of an inactive acyl-enzyme intermediate at 1738 ± 4 cm−1 .; Ser70, the active site nucleophile, was mutated to an alanine to make stable Michaelis complexes with various substrates. Comparison of the β-lactam carbonyl stretching frequency in the free and enzyme-bound substrate revealed an average decrease of 13 cm−1, indicating substantial strain/distortion of the carbonyl in the ES complex. The carbonyl's vibrational frequency in the ES was consistently 1755 ± 2 cm−1, suggesting that the active site environment induces a similar conformation on the substrate. Using deuterium substitution, it was determined that the “oxyanion hole”, which involves hydrogen bonding to two backbone amides, is the major source of the enzyme-induced polarization.; FTIR was also used to determine whether acylation or deacylation was rate-limiting for Lys73 and Glu166 mutants. For K73A and K73A/K234A β-lactamase, acylation was the rate-limiting step with cephalosporins, whereas deacylation was rate-limiting for penicillins. The results demonstrate that Lys73 is important in both acylation and deacylation, but do not indicate that it functions as a general base. Conversely, E166A β-lactamase resulted in rate-limiting deacylation for both penicillins and cephalosporins. Nonetheless, acylation was impaired for this mutant, especially with cefoxitin. These results show that Glu166 is the general base in deacylation and also suggest that this residue is responsible for activating Ser70 in acylation. Furthermore, K73A, K73A/K234A and E166A β-lactamase favored a branched-pathway mechanism and the formation of the inactive acyl-enzyme, when deacylation was rate-limiting.