ENDOR Structural Characterization of a Catalytically Competent Acylenzyme Reaction Intermediate of Wild-Type TEM-1 β-Lactamase Confirms Glutamate-166 as the Base Catalyst

Abstract

The catalytically competent active-site structure of a true acylenzyme reaction intermediate of TEM-1 beta-lactamase formed with the kinetically specific spin-labeled substrate 6-N-(2,2,5,5-tetramethyl-1-oxypyrrolinyl-3-carboxyl)-penicillanic acid isolated under cryoenzymologic conditions has been determined by angle-selected electron nuclear double resonance (ENDOR) spectroscopy. Cryoenzymologic experiments with use of the chromophoric substrate 6-N-[3-(2-furanyl)-propen-2-oyl]-penicillanic acid showed that the acylenzyme reaction intermediate could be stabilized in the -35 to -75 degrees C range with a half-life suitably long to allow freeze-quenching of the reaction species for ENDOR studies while a noncovalent Michaelis complex could be optically identified at temperatures only below -70 degrees C. The wild-type, Glu166Asn, Glu240Cys, and Met272Cys mutant forms of the mature enzyme were overexpressed in perdeuterated minimal medium to allow detection and assignment of proton resonances specific for the substrate and chemically modified amino acid residues in the active site. From analysis of the dependence of the ENDOR spectra on the setting of the static laboratory magnetic field H0, the dipolar contributions to the principal hyperfine coupling components were estimated to calculate the separations between the unpaired electron of the nitroxyl group and isotopically identified nuclei. These electron-nucleus distances were applied as constraints to assign the conformation of the substrate in the active site and of amino acid side chains by molecular 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 mutant. On the basis of the X-ray structure of the enzyme, the ENDOR distance constraints placed this water molecule within hydrogen-bonding distance to the carboxylate side chain of glutamate-166 as if it were poised for nucleophilic attack of the scissile ester bond. The ENDOR results provide experimental evidence of glutamate-166 in its functional role as the general base catalyst in the wild-type enzyme for hydrolytic breakdown of the acylenzyme reaction intermediate of TEM-1 beta-lactamase.

Structural and Biochemical Evidence That a TEM-1 β-Lactamase N170G Active Site Mutant Acts via Substrate-assisted Catalysis

“…The serine ␤-lactamase mechanism is similar to that of serine proteases (44), in that a proton is abstracted from the side chain oxygen of serine that acts a nucleophile and attacks the amide bond of the ␤-lactam ring (14,15,(17)(18)(19)(20)(21)24). An acylintermediate is then formed, and a highly ordered water molecule is activated for deacylation to hydrolyze the ␤-lactam and return the enzyme to its native state (20,22,(27)(28)(29).…”

Overexpression and Biosynthetic Deuterium Enrichment of TEM-1 β-Lactamase for Structural Characterization by Magnetic Resonance Methods

“…In Fig. 3, the resonances identified in the spectrum of the perdeuterated wt acylenzyme belong to the H(6) nucleus of the protiated penicilloyl moiety (9,31,46). Because the reaction intermediate of the E166N mutant in each case was prepared under the same conditions of temperature and pH or pD, it is seen that with increasing deuteration of the protein and solvent, elimination of overlapping proton resonances of the protein and solvent is achieved, gradually revealing the resonances of the (protiated) substrate.…”

“…The cryosolvent for the enzyme was 50:50 (v/v) methanol: H 2 O. Procedures for preparation of the acylenzyme reaction intermediate for spectroscopic studies are given elsewhere (9).…”

“…37 h, estimated on the basis of the rate constant governing deacylation (35), reaction of the E166N mutant with substrate also results essentially in a kinetically nonturnover condition. This situation can be approximated with the catalytically competent, wt enzyme only by application of cryoenzymologic conditions (9,(47)(48)(49). In ENDOR spectroscopy the shift of each resonance feature from the Larmor frequency is a direct function of the distance between the unpaired electron of the paramagnetic nitroxyl group (50) and the corresponding class of magnetic nuclei giving rise to the resonance feature through hyperfine coupling (51).…”

Spectroscopic analysis and docking simulation on the recognition and binding of TEM-1 β-lactamase with β-lactam antibiotics

“…Furthermore, a higher K MSV indicates that the chromophoric residues on the surface of the enzyme molecule are more readily accessible to KI, leading to greater quenching (42,43). The results in Table V demonstrated that, for the three antibiotics, all the quenching constants K MSV of TEM-1-antibiotic complexes are greater than those of TEM-1 β-lactamase, indicating that the chromophoric residues on the surface of TEM-1-antibiotic complexes are more accessible to KI than those on the surface of the enzyme molecule.…”