Effects of sulphate and urea on the stability and reversible unfolding of beta-lactamase from Staphylococcus aureus. Implications for the folding pathway of beta-lactamase.


Abstract

The reversible denaturation by urea of beta-lactamase from Staphylococcus aureus was followed in the presence and absence of ammonium sulphate by circular dichroism studies, difference absorption spectroscopy and measurement of enzyme activity. The multiple unfolding and refolding transitions demonstrate the existence of a thermodynamically stable state of intermediate conformation in equilibrium with the native (N) and fully unfolded (U) states. Its physical properties show that it is identical to the state H found on denaturation by guanidinium chloride. State H is 10.1 (+/-1.5) kJ mol-1 less stable than the native state and 10.1 (+/-1.6) kJ mol-1 more stable than the unfolded state. Ammonium sulphate shifts both the N in equilibrium H and H in equilibrium U transitions to concentrations of urea higher by 5.3 M per mole of sulphate. It has markedly different effects on the thermodynamic stabilities of states N and H, making delta G'N-H, O and delta G'H-U, O more negative by 41 kJ mol and 20 kJ mole, respectively, per mole of ammonium sulphate. The change in equilibrium constant for the N-H transition is reflected almost exclusively in a dramatic change of the unfolding rate constant, which is decreased by a factor of 10(11) on addition of 1.4 M-sulphate. The presence of the substrate benzyl penicillin has little effect on the equilibria or kinetics of the N-H transition. The results are discussed in terms of the nature of the N-H transition and of the ordering of intermediate states on the folding pathway. Study holds ProTherm entries: 10542, 10543, 10544, 10545, 10546, 10547 Extra Details: additive : EDTA(1 mM),transition 1 enzyme activity; ammonium sulphate; equilibrium constant;,folding pathway

Submission Details

ID: mfk7wa2R3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:40 p.m.

Version: 1

Publication Details
Mitchinson C;Pain RH,J. Mol. Biol. (1985) Effects of sulphate and urea on the stability and reversible unfolding of beta-lactamase from Staphylococcus aureus. Implications for the folding pathway of beta-lactamase. PMID:3875732
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
1DJC 1997-03-12 2.0 STRUCTURE OF BETA-LACTAMASE PRECURSOR, S70A MUTANT, AT 120K
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
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
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
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
1KGG 1999-05-28 2.3 STRUCTURE OF BETA-LACTAMASE GLU166GLN:ASN170ASP MUTANT
1GHI 2001-04-04 2.3 STRUCTURE OF BETA-LACTAMASE GLU166ASP:ASN170GLN MUTANT
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