ENDOR structural characterization of a catalytically competent acylenzyme reaction intermediate of wild-type TEM-1 beta-lactamase confirms glutamate-166 as the base catalyst

The catalytically competent active-site structure of a true acylenzyme reac tion intermediate of TEM-1 beta -lactamase formed with the kinetically spec ific spin-labeled substrate 6-N-(2,2,5,5-tetramethyl- 1-oxypyrrolinyl-3-car boxyl)-penicillanic acid isolated under cryoenzymologic conditions has been determined by angle-selected electron nuclear double resonance (ENDOR) spe ctroscopy. Cryoenzymologic experiments with use of the chromophoric substra te 6-N-[3-(2-furanyl)-propen-2-oyl]-penicillanic acid showed that the acyle nzyme reaction intermediate could be stabilized in the -35 to -75 degreesC range with a half-life suitably long to allow freeze-quenching of the react ion species for ENDOR studies while a noncovalent Michaelis complex could b e optically identified at temperatures only below -70 degreesC. The wild-ty pe, Glu 166Asn, Glu240Cys, and Met272Cys mutant forms of the mature enzyme were overexpressed in perdeuterated minimal medium to allow detection and a ssignment of proton resonances specific for the substrate and chemically mo dified amino acid residues in the active site. From analysis of the depende nce of the ENDOR spectra on the setting of the static laboratory magnetic f ield Ho, the dipolar contributions to the prillcipal hyperfine coupling com ponents were estimated to calculate the separations between the unpaired el ectron of the nitroxyl group and isotopically identified nuclei. These elec tron-nucleus distances were applied as constraints to assign the conformati on of the substrate in the active site and of amino acid side chains by mol ecular modeling. Of special interest was that the ENDOR spectra revealed a water molecule sequestered in the active site of the acylenzyme of the wild -type protein that was not detected in the deacylation impaired Glu166Asn m utant. On the basis of the X-ray structure of the enzyme, the ENDOR distanc e constraints placed this water molecule within hydrogen-bonding distance t o the carboxylate side chain of glutamate-166 as if it were poised for nucl eophilic attack of the scissile ester bond. The ENDOR results provide exper imental evidence of glutamate-166 in its functional role as the general bas e catalyst in the wild-type enzyme for hydrolytic breakdown of the acylenzy me reaction intermediate of TEM-1 beta -lactamase.