DIRECT NMR EVIDENCE FOR SUBSTRATE-INDUCED CONFORMATIONAL-CHANGES IN ABETA-LACTAMASE

Cefoxitin and other beta-lactam antibiotics with a methoxy group on th e alpha-face behave as very poor substrates of the Bacillus lichenifor mis beta-lactamase. The kinetic properties of the enzyme-cefoxitin sys tem made it theoretically suitable for a detailed structural study of the acyl-enzyme. Unfortunately, soaking the crystals in cefoxitin solu tion did not allow detection of a crystalline acyl-enzyme complex. In contrast, direct observation by n.m.r. of the stable acyl-enzyme forme d with cefoxitin and moxalactam indicated clear modifications of the e nzyme structure, which were reflected in the aromatic and high-field m ethyl regions of the spectrum. The return to the initial free enzyme s pectrum was concomitant with the hydrolysis of the acyl-enzyme, the pr ocess being slow enough to allow multidimensional n.m.r. experiments.