Differential scanning calorimetric study of the thermal unfolding of beta-lactamase I from Bacillus cereus.


Abstract

The irreversible thermal unfolding of the class A beta-lactamase I from Bacillus cereus has been investigated at pH 7.0, using differential scanning calorimetry (DSC) and inactivation kinetic techniques. DSC transitions showed a single peak with a denaturation enthalpy of 646 kJ.mol-1 and were moderately scan rate dependent, suggesting that the process was partially kinetically controlled. The inactivation kinetics at constant temperature showed that the irreversible denaturation of the enzyme occurs as the sum of two exponential terms whose amplitudes are strongly temperature dependent within the transition range so that, at the lowest temperatures within this interval, irreversible inactivation would proceed mainly through the slow phase. The fraction of irreversibly denatured enzyme (D) as a function of temperature for a given scanning rate was calculated by numerical integration of the kinetic equation with temperature, using previously determined kinetic parameters. This D form was the most populated of the unfolded states only at temperatures well above the maximum in the calorimetric transition. Combination of the results of kinetic and DSC experiments has allowed us to separate the contribution of the final D state to the excess enthalpy change from the contribution arising from the reversibly denatured forms of the enzyme (I(i), i = 1,..., n), with the resulting conclusion that the scan rate dependence of the calorimetric traces was the result of two different dynamic effects, viz., the irreversible step and a slow relaxation process during formation of the reversibly denatured intermediate states. Finally, the problems of using results obtained at a single scan rate to validate the two-state kinetic model are commented on. Study holds ProTherm entries: 4802 Extra Details: (i) scan rate is 20 C/h,(ii) reversibility is scanning-rate dependent thermogram inactivation kinetics; temperature dependent;,intermediate states; two-state kinetic model

Submission Details

ID: 3GCDTe79

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:27 p.m.

Version: 1

Publication Details
Arriaga P;Menéndez M;Villacorta JM;Laynez J,Biochemistry (1992) Differential scanning calorimetric study of the thermal unfolding of beta-lactamase I from Bacillus cereus. PMID:1633173
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
2M5D 2013-10-09 Solution Structure of the Bacillus cereus Metallo-Beta-Lactamase BcII in Complex with R-Thiomandelic Acid
2M5C 2013-10-09 Solution Structure of the Bacillus cereus Metallo-Beta-Lactamase BcII
5FQA 2016-08-10 1.1 Crystal Structure of Bacillus cereus Metallo-Beta-Lactamase II
4C1H 2014-08-27 1.1 Crystal structure of the metallo-beta-lactamase BCII with L-captopril
6F2N 2018-10-03 1.15 Crystal structure of BCII Metallo-beta-lactamase in complex with KDU197
6EUM 2018-10-03 1.18 CRYSTAL STRUCTURE OF BCII METALLO-BETA-LACTAMASE IN COMPLEX WITH DZ-307
4C1C 2014-08-27 1.18 Crystal structure of the metallo-beta-lactamase BCII with D-captopril
4C09 2014-08-20 1.2 Crystal structure of the metallo-beta-lactamase BCII
1MQO 2004-05-18 1.35 Metallo-beta-lactamase BcII Cd substituted from Bacillus cereus at 1.35 angstroms resolution
5JMX 2017-05-24 1.44 Crystal Structure of BcII metallo-beta-lactamase in complex with DZ-305
3I11 2009-12-29 1.45 Cobalt-substituted metallo-beta-lactamase from Bacillus cereus
6EWE 2018-10-03 1.46 Crystal structure of BCII Metallo-beta-lactamase in complex with DZ-308
3I15 2009-12-29 1.55 Cobalt-substituted metallo-beta-lactamase from Bacillus cereus: residue Cys168 fully oxidized
3I14 2009-12-29 1.55 Cobalt-substituted metallo-beta-lactamase from Bacillus cereus: residue Cys168 partially oxidized
3KNS 2010-11-24 1.58 Bacillus cereus metallo-beta-lactamase Cys221Asp mutant, 20 mM Zn(II)
3I0V 2009-12-29 1.6 Bacillus cereus metallo-beta-lactamase: apo form
4NQ4 2014-11-26 1.67 Bacillus cereus Zn-dependent metallo-beta-lactamase at pH 7
3BC2 1999-04-20 1.7 METALLO BETA-LACTAMASE II FROM BACILLUS CEREUS 569/H/9 AT PH 6.0, MONOCLINIC CRYSTAL FORM
2BC2 1999-04-20 1.7 METALLO BETA-LACTAMASE II FROM BACILLUS CEREUS 569/H/9 AT PH 6.0, TRIGONAL CRYSTAL FORM
3KNR 2010-11-24 1.71 Bacillus cereus metallo-beta-lactamase Cys221Asp mutant, 1 mM Zn(II)
3I13 2009-12-29 1.74 Bacillus cereus Zn-dependent metallo-beta-lactamase at pH 5.8
2BFL 2005-03-31 1.8 Bacillus cereus metallo-beta-lactamase (BcII) Arg (121) Cys mutant. Solved at pH5 using 20mM ZnSO4 in buffer. 1mM DTT was used as a reducing agent.
2NZE 2007-05-29 1.8 Structure of beta-lactamase II from Bacillus cereus. R121H, C221S double mutant. Space group P3121.
4TYT 2014-11-26 1.8 Crystal Structure of BcII metallo-beta-lactamase in complex with ML302F
4NQ6 2014-11-26 1.8 Bacillus cereus Zn-dependent metallo-beta-lactamase at pH 7 complexed with compound L-CS319
2NYP 2007-05-22 1.84 Structure of beta-lactamase II from Bacillus cereus. R121H, C221D doble mutant with two zinc ions.
1DXK 2000-08-25 1.85 Metallo-beta-lactamase from Bacillus cereus 569/H/9 C168S mutant
1BVT 1998-09-23 1.85 METALLO-BETA-LACTAMASE FROM BACILLUS CEREUS 569/H/9
5FQB 2016-08-10 1.9 Crystal Structure of Bacillus cereus Metallo-Beta-Lactamase with 2C
1BC2 1998-10-14 1.9 ZN-DEPENDENT METALLO-BETA-LACTAMASE FROM BACILLUS CEREUS
2UYX 2007-05-08 1.95 metallo-beta-lactamase (1BC2) single point mutant D120S
2BFK 2005-03-31 2.0 Bacillus cereus metallo-beta-lactamase (BcII) Arg (121) Cys mutant. Solved at pH7 using 20mM ZnSO4 in buffer. 1mM DTT was used as a reducing agent
2BG7 2005-03-31 2.1 Bacillus cereus metallo-beta-lactamase (BcII) Arg (121) Cys mutant. Solved at pH4.5 using 20 Micromolar ZnSO4 in the buffer. 1mM DTT was used as a reducing agent. Cys221 is oxidized.
5W8W 2017-07-12 2.25 Bacillus cereus Zn-dependent metallo-beta-lactamase at pH 7 - new refinement
2NZF 2007-05-29 2.28 Structure of beta-lactamase II from Bacillus cereus. R121H, C221S double mutant. Space group C2.
2NXA 2007-05-22 2.29 Structure of Zn-dependent Metallo-Beta-Lactamase from Bacillus Cereus R121H, C221D Double Mutant
4NQ5 2014-11-26 2.29 Bacillus cereus Zn-dependent metallo-beta-lactamase at pH 7 complexed with compound CS319
2BG6 2005-03-31 2.3 Bacillus cereus metallo-beta-lactamase (BcII) Arg (121) Cys mutant. Solved at pH5 using 20 Micromolar ZnSO4 in the buffer. 1mM DTT was used as a reducing agent. Cys221 is oxidized.
2BFZ 2005-03-31 2.3 Bacillus cereus metallo-beta-lactamase (BcII) Arg (121) Cys mutant. Solved at pH4.5 using 20mM ZnSO4 in buffer. 1mM DTT was used as a reducing agent. Cys221 is oxidized.
2BG2 2005-03-31 2.4 Bacillus cereus metallo-beta-lactamase (BcII) Arg (121) Cys mutant. Solved at pH4.5 using 20mM ZnSO4 in the buffer. 1mM DTT and 1mM TCEP- HCl were used as reducing agents. Cys221 is reduced.
6DJA 2019-05-29 2.48 ZN-DEPENDENT 5/B/6 METALLO-BETA-LACTAMASE FROM BACILLUS CEREUS
1BMC 1996-08-28 2.5 STRUCTURE OF A ZINC METALLO-BETA-LACTAMASE FROM BACILLUS CEREUS
2BG8 2005-03-31 2.5 Bacillus cereus metallo-beta-lactamase (BcII) Arg (121) Cys mutant. Solved at pH4.5 using 20 Micromolar ZnSO4 in the buffer. 1mM DTT and 1mM TCEP-HCl were used as reducing agents.
2BGA 2005-03-31 2.7 Bacillus cereus metallo-beta-lactamase (BcII) Arg (121) Cys mutant. Solved at pH7 using 20 Micromolar ZnSO4 in the buffer. 1mM DTT was used as a reducing agent. Cys221 is oxidized.
3FCZ 2008-12-09 2.8 Adaptive protein evolution grants organismal fitness by improving catalysis and flexibility

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
94.5 Metallo-beta-lactamase type 2 P14488 BLAB_BACCE
93.6 Metallo-beta-lactamase type 2 P10425 BLA2_BAC17
100.0 Metallo-beta-lactamase type 2 P04190 BLA2_BACCE