Structure at the active site of an acylenzyme of alpha-chymotrypsin and implications for the catalytic mechanism. An electron nuclear double resonance study.

Wells, Gregg B.; Mustafi, Devkumar; Makinen, Marvin W.

doi:10.1016/s0021-9258(17)41816-3

Cited by 14 publications

(11 citation statements)

References 65 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The penicilloyl moieties were then superposed so as to remove steric overlap with active-site residues while placing the 2-carboxylate group of the substrate into the anion binding site and the β-lactam CdO group into the oxyanion hole as in the Glu166Asn acylenzyme. Aside from the methyl ester group and the cleaved β-lactam bond, the structure of the spin-labeled methyl penicilloate ester differs from that of SLPEN primarily through the tetrahedral valence angles around C(6) and the value of the τ[N(1)-C( 5)-C(6)-C (7)] torsion angle (12).…”

Section: Methodsmentioning

confidence: 99%

“…We can then use the observed hfc components directly to estimate electron-nucleus distances. The validity of this approximation has been demonstrated in numerous ENDOR studies of spin-labeled compounds, including spin-labeled penicillin and cephalosporin (7)(8)(9)(10)(11)(12)(44)(45)(46)(47).…”

Section: Endor Characterization Of the Acylenzyme Of Tem-1 β-Lactamasementioning

confidence: 96%

“…The effective dipolar position of the unpaired electron spin associated with the nitroxyl group lies approximately at the midpoint of the N-O bond and is defined to within (0.04 Å (9,45). By application of ENDOR we have shown that dipolar electron-nucleus distances can be determined over a 4-10 Å range with errors no greater than 5% based on ENDOR line width (7)(8)(9)(10)(11)(12)(44)(45)(46). This precision of structural analysis is greater than that achieved by other physical methods for molecules in solution and in fact is exceeded only by that of single-crystal X-ray diffraction.…”

Section: Endor Characterization Of the Acylenzyme Of Tem-1 β-Lactamasementioning

confidence: 96%

“…We have shown through a series of studies published from this laboratory that application of electron nuclear double resonance (ENDOR) spectroscopy for structure analysis of true, catalytically competent intermediates of enzyme catalyzed reactions provides an incisive means to identify and structurally characterize enzyme:substrate interactions that are catalytically important and are not observed in enzyme−inhibitor complexes ( − ). We have applied this approach to characterize the acylenzyme reaction intermediate of TEM-1 β-lactamase, using a kinetically specific, nitroxyl spin-labeled penicillin ( , ) as a paramagnetic probe of active-site structure.…”

mentioning

confidence: 99%

See 3 more Smart Citations

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

2001

View full text Add to dashboard Cite

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.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Endor Characterization Of the Acylenzyme Of Tem-1 β-Lactamasementioning

confidence: 96%

Section: Endor Characterization Of the Acylenzyme Of Tem-1 β-Lactamasementioning

confidence: 96%

mentioning

confidence: 99%

See 2 more Smart Citations

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

2001

View full text Add to dashboard Cite

show abstract

“…We have shown that this hf coupling can be measured accurately by ENDOR to obtain electron−nucleus dipolar distances in the range of 3−11 Å with errors as low as 5% based on ENDOR line widths. In these studies, we have shown that ENDOR of nitroxyl spin- labels in combination with molecular modeling provides a general method for the determination of the structure and conformation of both small molecules and enzyme reaction intermediates in solution with a precision that is exceeded only by that of single-crystal X-ray diffraction ( − ).…”

mentioning

confidence: 99%

Structure of Spin-Labeled Methylmethanethiolsulfonate in Solution and Bound to TEM-1 β-Lactamase Determined by Electron Nuclear Double Resonance Spectroscopy

et al. 2001

View full text Add to dashboard Cite

Site-directed spin-labeling of proteins whereby the spin-label methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl)methanethiolsulfonate (SLMTS) is reacted with the -SH groups of cysteinyl residues incorporated into a protein by mutagenesis has been successfully applied to investigate secondary structure and conformational transitions of proteins. In these studies, it is expected that the spin-label moiety adopts different conformations dependent on its local environment. To determine the conformation of SLMTS in solution reacted with L-cysteine (SLMTCys) and bound in the active site of the Glu240Cys mutant of TEM-1 beta-lactamase, we have synthesized SLMTS both of natural abundance isotope composition and in site-specifically deuterated forms for electron nuclear double resonance (ENDOR) studies. ENDOR-determined electron-proton distances from the unpaired electron of the nitroxyl group of the spin-label to the methylene and methyl protons of SLMTS showed three conformations of the oxypyrrolinyl ring with respect to rotation around the S-S bond dependent on the solvent dielectric constant. For SLMTCys, two conformations of the molecule were compatible with the ENDOR-determined electron-nucleus distances to the side-chain methylene protons and to H(alpha) and H(beta1,2) of cysteine. To determine SLMTS conformation reacted with the Glu240Cys mutant of TEM-1 beta-lactamase, enzyme was overexpressed in both ordinary and perdeuterated minimal medium. Resonance features of H(alpha) and H(beta1,2) of the Cys240 residue of the mutant and of the side-chain methylene protons within the spin-label moiety yielded electron-proton distances that sterically accommodated the two conformations of free SLMTCys in solution.

show abstract

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

Sosa‐Peinado

Mustafi

Makinen

2000

Protein Expression and Purification

View full text Add to dashboard Cite

Structure at the active site of an acylenzyme of alpha-chymotrypsin and implications for the catalytic mechanism. An electron nuclear double resonance study.

Cited by 14 publications

References 65 publications

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

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

Structure of Spin-Labeled Methylmethanethiolsulfonate in Solution and Bound to TEM-1 β-Lactamase Determined by Electron Nuclear Double Resonance Spectroscopy

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

Contact Info

Product

Resources

About