Direct n.m.r. evidence for substrate-induced conformational changes in a beta-lactamase.
Cefoxitin and other beta-lactam antibiotics with a methoxy group on the alpha-face behave as very poor substrates of the Bacillus licheniformis beta-lactamase. The kinetic properties of the enzyme-cefoxitin system made it theoretically suitable for a detailed structural study of the acyl-enzyme. Unfortunately, soaking the crystals in cefoxitin solution did not allow detection of a crystalline acyl-enzyme complex. In contrast, direct observation by n.m.r. of the stable acyl-enzyme formed with cefoxitin and moxalactam indicated clear modifications of the enzyme structure, which were reflected in the aromatic and high-field methyl regions of the spectrum. The return to the initial free enzyme spectrum was concomitant with the hydrolysis of the acyl-enzyme, the process being slow enough to allow multidimensional n.m.r. experiments.
Laboratoire d'Enzymologie, Université de Liège, Belgium.
M
M
Jamin
C
C
Damblon
A M
AM
Bauduin-Misselyn
F
F
Durant
G C
GC
Roberts
P
P
Charlier
G
G
Llabres
J M
JM
Frère
D000900:Anti-Bacterial Agents; D001407:Bacillus / Q000201:enzymology; D002440:Cefoxitin; D002942:Circular Dichroism; D003461:Crystallography; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism
D000900:Anti-Bacterial Agents; D002440:Cefoxitin; D001618:beta-Lactamases