We have synthesized spin-labeled analogs of cephalosporin and penicillin and of their solvolytic methyl ester products for structural characterization by electron nuclear double resonance (ENDOR) spectroscopy. With beta-lactamase I of Bacillus cereus 569/H/9 and the metallo-beta-lactamase of Bacillus cereus 5/B/6, the spin-labeled penicillin and cephalosporin were shown to be as kinetically specific and catalytically active as the classical substrates benzylpenicillin and cephalosporin-C, respectively. Conformations of the spin-labeled antibiotics and of their methanolysis products have been compared on the basis of structure determination by ENDOR and molecular modeling. While the conformation of spin-labeled penicillin has been shown previously to be identical to that of amoxycillin (Mustafi, D.; Makinen, M. W. J. Am. Chem. Soc. 1995, 117, 6739-6746), the ENDOR-assigned conformation of spin-labeled cephalosporin is essentially identical to the X-ray-defined structure of cephaloglycine (Sweet, R. M.; Dahl, L. F. J. Am. Chem. Soc. 1970, 92, 5489-5507). We have also determined by ENDOR the location of a methanol molecule that is hydrogen-bonded to the pseudopeptide NH group of the spin-labeled cephalosporin. The ENDOR results assign the location of the methanol hydroxyl group on the sterically hindered endo side of the B-lactam ring. This position suggests that the hydroxyl group of the methanol molecule is poised for nucleophilic attack on the carbonyl carbon of the beta-lactam ring. We have also compared the ENDOR-determined structures of the free beta-lactam antibiotics and of their methanolysis products with the X-ray-defined structure of the benzylpenicilloyl acylenzyme intermediate of the E166N mutant of TEM-1 beta-lactamase (Strynadka et al, Nature 1992, 359, 700-705). The results showed that hydrolysis relieves geometrical strain in the fused beta-lactam ring, as measured by the [N(1)-C(6)-C(7)-C(8)] dihedral angle in cephalosporin or by the corresponding [N(1)-C(5)-C(6)-C(7)] dihedral angle in penicillin. While the value of this dihedral angle is eclipsed at similar to 0 degrees for the intact antibiotic substrates and staggered at -35 degrees for the benzylpenicilloyl intermediate, the methanolysis products exhibited trans conformations at -160 degrees. The results illustrate how hydrolysis of the beta-lactam bond in penicillin or cephalosporin antibiotics progresses favorably from a geometrically strained to a less strained conformation in the acylenzyme reaction intermediate and finally to an inherently conformationally stable product.