TABLE 3.

Kinetic parameters for the hydrolysis of β-lactam substrates by the wild-type (pBSKS/TEM-1) and mutant (Asp²⁷⁶) β-lactamases^a

Substrate	Wild-type (TEM-1)			Mutant (Asn276Asp)
	kcat (s−1)b	Km (μM)c	kcat/Km (μM−1 · s−1)	kcat (s−1)	Km (μM)	kcat/Km (μM−1 · s−1)
Benzylpenicillin	1,160	28	41.2	1,980	105	18.9
Amoxicillin	1,080	32	33.8	1,620	276	5.9
Ticarcillin	139	13	10.7	269	113	2.4
Piperacillin	1,060	67	15.9	1,660	232	7.2
Mezlocillin	1,280	68	18.8	2,100	347	6.0
Mecillinam	822	1,730	0.48	436	793	0.55
Imipenem	—d	NDe	ND	—	198f	ND
Cephalothin	312	341	0.9	273	389	0.7
Cephaloridine	1,630	1,020	1.6	1,830	397	4.6
Ceftazidime	—	ND	ND	—	261f	ND
Cefoperazone	413	402	1.68	513	405	1.3

^aThe k_cat and K_m of Asn276Asp for oxacillin, aztreonam, cefotaxime, ceftriaxone, cefoxitine, and cefuroxime could not be measured (there was no detectable hydrolysis or V_max was <2% and K_i wash >500 μM). 

^bThe standard deviation s for the analysis were ±5%. 

^cThe standard deviation s were ±10% for K_m values of <100 μM and ±20% for K_m values of >100 μM. 

^d—, no detectable hydrolysis. 

^eND, not determined. 

^fK_m was determined as K_i by the reporter substrate method. For more details, see Materials and Methods.