Characterization of covalently bound enzyme inhibitors as transition-state analogs by protein stability measurements: phosphonate monoester inhibitors of a beta-lactamase.


Abstract

An experimental method is described for determining whether a covalent enzyme-inhibitor complex has the properties expected of a transition-state analog. The method involves a comparison of the noncovalent interaction energies between the enzyme and the inhibitor on one hand (determined from protein denaturation thermodynamics) and the analogous transition state on the other (determined from kinetic measurements). These two quantities should presumably be large (in comparison with the interaction energies of substrates or reaction intermediates) and close to equal for a good transition state analog; the former is seen dramatically in a large increase in protein stability. The method is absolute in the sense that it does not require a crystal structure of the inhibited enzyme or any preconceptions as to the mechanism of action of the enzyme except those which led to adoption of the potential transition state analog and which might turn out to be right or wrong. In this paper the method is quantitatively applied to the inhibition of the Staphylococcus aureus PC1 beta-lactamase by phosphonate monoesters. It is concluded that the enzyme-inhibitor complex in this case is likely to be a good transition-state mimic. Therefore, mechanistic interpretation of the crystal structure of the complex can be made with more confidence. A semiquantitative assessment of the situation with serine proteinases is also made. It is concluded, in agreement with predictions based on the generally accepted mechanism and on crystal structures, that anionic, but not neutral, phosph(or/on)yl derivatives are good transition-state analogs. Study holds ProTherm entries: 4647 Extra Details: enzyme inhibitors; transition-state analog; serine proteinases;,noncovalent interaction energies

Submission Details

ID: YpWBaJij

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:26 p.m.

Version: 1

Publication Details
Rahil J;Pratt RF,Biochemistry (1994) Characterization of covalently bound enzyme inhibitors as transition-state analogs by protein stability measurements: phosphonate monoester inhibitors of a beta-lactamase. PMID:8286328
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1GHP 2001-04-04 1.76 STRUCTURES OF THE ACYL-ENZYME COMPLEX OF THE STAPHYLOCOCCUS AUREUS BETA-LACTAMASE MUTANT GLU166ASP:ASN170GLN WITH DEGRADED BENZYLPENICILLIN
1ALQ 1997-09-26 1.8 CIRCULARLY PERMUTED BETA-LACTAMASE FROM STAPHYLOCOCCUS AUREUS PC1
1GHM 2001-04-04 1.86 Structures of the acyl-enzyme complex of the staphylococcus aureus beta-lactamase mutant GLU166ASP:ASN170GLN with degraded cephaloridine
1DJA 1997-03-12 1.9 STRUCTURE OF BETA-LACTAMASE PRECURSOR, K73H MUTANT, AT 298K
1KGE 1997-04-01 2.0 STRUCTURE OF BETA-LACTAMASE ASN 170 MET MUTANT
3BLM 1991-01-15 2.0 REFINED CRYSTAL STRUCTURE OF BETA-LACTAMASE FROM STAPHYLOCOCCUS AUREUS PC1 AT 2.0
1DJC 1997-03-12 2.0 STRUCTURE OF BETA-LACTAMASE PRECURSOR, S70A MUTANT, AT 120K
1DJB 1997-03-12 2.1 STRUCTURE OF BETA-LACTAMASE PRECURSOR, S70A MUTANT, AT 298K
1KGF 1997-03-12 2.2 STRUCTURE OF BETA-LACTAMASE ASN 170 GLN MUTANT
1BLC 1994-01-31 2.2 INHIBITION OF BETA-LACTAMASE BY CLAVULANATE: TRAPPED INTERMEDIATES IN CRYOCRYSTALLOGRAPHIC STUDIES
1BLP 1994-04-30 2.3 STRUCTURAL BASIS FOR THE INACTIVATION OF THE P54 MUTANT OF BETA-LACTAMASE FROM STAPHYLOCOCCUS AUREUS PC1
1BLH 1994-08-31 2.3 STRUCTURE OF A PHOSPHONATE-INHIBITED BETA-LACTAMASE. AN ANALOG OF THE TETRAHEDRAL TRANSITION STATE(SLASH)INTERMEDIATE OF BETA-LACTAM HYDROLYSIS
1GHI 2001-04-04 2.3 STRUCTURE OF BETA-LACTAMASE GLU166ASP:ASN170GLN MUTANT
1KGG 1999-05-28 2.3 STRUCTURE OF BETA-LACTAMASE GLU166GLN:ASN170ASP MUTANT
1OME 1998-05-27 2.3 CRYSTAL STRUCTURE OF THE OMEGA LOOP DELETION MUTANT (RESIDUES 163-178 DELETED) OF BETA-LACTAMASE FROM STAPHYLOCOCCUS AUREUS PC1
1PIO 1996-03-08 2.8 AN ENGINEERED STAPHYLOCOCCUS AUREUS PC1 BETA-LACTAMASE THAT HYDROLYSES THIRD GENERATION CEPHALOSPORINS

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Beta-lactamase P00807 BLAC_STAAU