Enzyme-induced strain/distortion in the ground-state ES complex in beta-lactamase catalysis revealed by FTIR

Class A beta-lactamases hydrolyze penicillins and other beta-lactams via an acyl-enzyme catalytic mechanism. Ser70 is the active site nucleophile. By constructing the S70A mutant, which is unable to form the acyl-enzyme inter mediate, it was possible to make stable ES complexes with various substrate s. The stability of such Michaelis complexes permitted acquisition of their infrared spectra. Comparison of the beta-lactam carbonyl stretch frequency (vco) in the foe and enzyme-bound substrate revealed an average decrease o f 13 cm(-1), indicating substantial strain/distortion of the lactam carbony l when bound in the ES complex. Interestingly, regardless of the frequency of the C=O stretch in the free substrate, when complexed to Bacillus lichen iformis beta-lactamase, the frequency was always 1755 +/- 2 cm(-1). This su ggests the active site environment induces a similar conformation of the be ta-lactam in all substrates when bound to the enzyme. Using deuterium subst itution, it was shown that the "oxyanion hole", which involves hydrogen bon ding to two backbone amides, is the major source of the enzyme-induced stra in/distortion. The very weak catalytic activity of the S70A beta-lactamase suggests enzyme-facilitated hydrolysis due to substrate distortion on bindi ng to the enzyme. Thus the binding of the substrate in the active site indu ces substantial strain and distortion that contribute significantly to the overall rate enhancement in beta-lactamase catalysis.