1. Volume 8, Issue 8, Pages 831-842 (January 2001)
Quantitative analysis of the stabilization by substrate of Staphylococcus aureus PC1 β- lactamase 
Annabelle Lejeune, Marc Vanhove, Josette Lamotte-Brasseur, Roger H. Pain, Jean-Marie Frère, André Matagne 
Chemistry & Biology 
Volume 8, Issue 8, Pages (January 2001)
DOI: /S (01)
Copyright © 2001 Elsevier Ltd Terms and Conditions

2. Fig. 1
Thermal unfolding transition of S. aureus PC1 β-lactamase determined by measurements of intrinsic fluorescence (○) and catalytic activity (•). Data have been normalized such that the value of the native state is 1. Both sets of data were analyzed on the basis of a two-state model and the lines represent the best fit to Eq. 1, calculated using Tm=41.8±0.3°C and 41.2±0.6°C, and ΔHm=420±30 kJ mol−1 and 300±30 kJ mol−1, for fluorescence and activity measurements, respectively. The latter were performed with 1 mM benzylpenicillin, as described in Section 4.
Chemistry & Biology 2001 8, DOI: ( /S (01) )
Copyright © 2001 Elsevier Ltd Terms and Conditions

3. Fig. 2
The rate of thermal inactivation of S. aureus PC1 β-lactamase in the presence of cefazolin. The enzyme (0.05 μM) was incubated at 52°C with 68 μM cefazolin in 50 mM sodium phosphate buffer, pH 7. Hydrolysis of the reporter substrate was monitored by recording the decrease in absorbance at 260 nm and, according to the method described in [46], an apparent first-order rate constant (ku) value of 0.012±0.001 s−1 was computed. The curve represents the best fit of the data to Eq. 6, modified as in [46]. Note that spontaneous hydrolysis of cefazolin occurs throughout the time-course of the experiment. However, this phenomenon does not account for more than 8% of substrate utilization and can thus be considered as occurring at a constant rate. It only influences the v0 and vss values, and not ku. After correction for this phenomenon, v0=5.73 μmol min−1 mg−1 and vss=0.24 μmol min−1 mg−1. The inset shows the simulated kinetics (see text).
Chemistry & Biology 2001 8, DOI: ( /S (01) )
Copyright © 2001 Elsevier Ltd Terms and Conditions

4. Fig. 3
Influence of the concentration of cefazolin on the inactivation rate (ku) of the S. aureus PC1 β-lactamase at 52°C. • Experimental data. These values were obtained as described in Section 4 (see also Fig. 2). ○ Simulated data computed on the basis of model 2, as described in the text. Values given in Table 3 were used for kcat, Km, k2, k3, K′, ku,1 and kf,1, with ku,2=3×10−3 s−1, kf,2=0.039 M−1s−1, ku,3=3×10−3 s−1 and kf,3=1.5×10−3 s−1.
Chemistry & Biology 2001 8, DOI: ( /S (01) )
Copyright © 2001 Elsevier Ltd Terms and Conditions

5. Fig. 4
Temperature dependence of the turnover number (kcat) for S. aureus PC1 β-lactamase and cefazolin, shown as an Arrhenius plot. kcat has units of s−1 and T has units of K. (•) Measured values; (○) extrapolated value at 52°C (kcat=2.1 s−1). Errors are given as standard deviations. The continuous line is the best fit to ln kcat=ln A−Ea/RT, with Ea=82±2 kJ mol−1.
Chemistry & Biology 2001 8, DOI: ( /S (01) )
Copyright © 2001 Elsevier Ltd Terms and Conditions

6. Fig. 5
Temperature dependence of Km for S. aureus PC1 β-lactamase and cefazolin. Km has units of μM. (•) Measured values; (○) value at 52°C (Km≈13 μM) based on linear extrapolation. Errors are given as standard deviations.
Chemistry & Biology 2001 8, DOI: ( /S (01) )
Copyright © 2001 Elsevier Ltd Terms and Conditions

7. Fig. 6
Kinetics of interaction between S. aureus β-lactamase and cefazolin at 22.5°C. Variation of ka (=k3+(k2x[S])/(K′+[S])) with cefazolin concentration. (•) Stopped-flow measured values; (○) kcat (see text). The data were fitted (full line) to Eq. 8, yielding k2=0.92±0.02 s−1 and K′=13.5±0.8 μM.
Chemistry & Biology 2001 8, DOI: ( /S (01) )
Copyright © 2001 Elsevier Ltd Terms and Conditions

8. Fig. 7
Temperature dependence of the acylation rate constant (k2) for S. aureus PC1 β-lactamase and cefazolin, shown as an Arrhenius plot. k2 has units of s−1 and T has units of K. (•) Measured values; (○) extrapolated value at 52°C (k2=45 s−1). Errors (S.D. values) on these measurements are <10%. The continuous line is the best fit to ln k2=ln A−Ea/RT, with Ea=103±5 kJ mol−1.
Chemistry & Biology 2001 8, DOI: ( /S (01) )
Copyright © 2001 Elsevier Ltd Terms and Conditions

9. Fig. 8
Temperature dependence of the equilibrium constant (Ku,1) and of the microscopic rate constants for unfolding (ku,1) and refolding (kf,1) of the free S. aureus PC1 β-lactamase. Ku,1 has no units, ku, ku,1 and kf,1 have units of s−1, and T has units of K. (a) van’t Hoff plot of Ku,1=ku,1/kf,1. The continuous line is the best fit to ln K=−ΔH/RT+ΔS/R, with ΔH=425±10 kJ mol−1 and ΔS=1.35±0.02 kJ mol−1 K−1. (b) Arrhenius plot of ku=ku,1+kf,1. The continuous line is the best fit to ln ku=ln A−Ea/RT, with Ea=210±3 kJ mol −1. (c) Arrhenius plot of ku,1. The continuous line is the best fit to ln ku,1=ln A−Ea/RT, with Ea=250±10 kJ mol −1. Note that values at 52°C, 55°C and 60°C were determined experimentally (ku,1=ku). (d) Arrhenius plot of kf,1. The continuous line is the best fit to Eq. 4. The activation parameters derived from this fit are ΔGUN#=95.4 kJ mol−1, ΔSi#=2.1 kJ mol−1 K−1 and ΔCpi#=−33.3 kJ mol−1 K−1. The extrapolated kf,1 (2.2×10−4 s−1) value at 52°C is represented by an open circle.
Chemistry & Biology 2001 8, DOI: ( /S (01) )
Copyright © 2001 Elsevier Ltd Terms and Conditions

10. Fig. 9
Simulated time-courses at 52°C of the native and unfolded enzyme species in the presence of 68 μM cefazolin. Model 2 and Eqs. 9–17 were used, with the parameter values given in Table 3 and in the legend of Fig. 3. The continuous line corresponds to x (=E+ES+ES*), the dashed line to y (=E′) and the dotted line to z (=E′S*).
Chemistry & Biology 2001 8, DOI: ( /S (01) )
Copyright © 2001 Elsevier Ltd Terms and Conditions

11. Fig. 10
Measured (filled bars) and calculated (empty bars) vss/v0 ratios as a function of cefazolin concentration. vss/v0 ratios were calculated on the basis of model 2, with the parameter values given in Table 3 and in the legend of Fig. 3.
Chemistry & Biology 2001 8, DOI: ( /S (01) )
Copyright © 2001 Elsevier Ltd Terms and Conditions

12. Chemistry & Biology 2001 8, 831-842DOI: (10
Chemistry & Biology 2001 8, DOI: ( /S (01) )
Copyright © 2001 Elsevier Ltd Terms and Conditions

13. Chemistry & Biology 2001 8, 831-842DOI: (10
Chemistry & Biology 2001 8, DOI: ( /S (01) )
Copyright © 2001 Elsevier Ltd Terms and Conditions