Table 1. Kinetic parameters for the hydrolysis of different β-lactam substrates by WT BcII and mutant M5.

	BcII			M5			M5/BcII ratio
Substrate	kcat, s-1	KM, μM	kcat/KM, M-1·s-1	kcat, s-1	KM, μM	kcat/KM, M-1·s-1	kcat	KM	kcat/KM
Imipenem	279	687	4.06 × 105	129	721	1.79 × 105	0.462	1.05	0.440
Benzylpenicillin	1020	662	1.54 × 106	570	574	9.93 × 105	0.559	0.867	0.644
Nitrocefin	30.1	9.82	3.07 × 106	81.3	27.8	2.92 × 106	2.70	2.83	0.954
Cefotaxime	80.5	43.3	1.86 × 106	44.9	30.3	1.49 × 106	0.557	0.699	0.796
Cephaloglycin	ND	ND	1.89 × 106	ND	ND	1.80 × 106	ND	ND	0.952
Cephalexin	3.32	125	2.65 × 104	41.2	209	1.97 × 105	12.4	1.67	7.42
Cefaclor	73.5	328	2.24 × 105	410	422	9.71 × 105	5.58	1.28	4.35
Cephadroxyl	19.2	789	2.43 × 104	75.2	200	3.76 × 105	3.91	0.253	15.4
Cephradine	ND	ND	6.81 × 104	ND	ND	3.51 × 105	ND	ND	5.15
Ceftazidime	13.1	426	3.07 × 104	24.4	262	9.31 × 104	1.86	0.615	3.03
Cephaloridine	57.9	1250	4.63 × 104	68.1	204	3.34 × 105	1.17	0.163	7.17

Conditions: 10 mM Hepes, 0.2 M NaCl, 50 μg/ml BSA, and 20 μM Zn (pH 7.5) at 30°C. Reported values are the average of at least three independent determinations. Standard deviation values were <5%. ND, not determined