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


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 UniProtKB Entries

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