TABLE 2.

Branched pathway kinetics for cefotaxime and ceftazidime hydrolysis by TEM-1 β-lactamase mutants

CAZ, ceftazidime; CTX, cefotaxime; AS, ammonium sulfate.

TEM-1 mutant	Substrate	Branched pathway parametersa			Burstb
		vi	vss	k
		μmol/s	μmol/s	s−1
E166Y	CAZ
	0% AS	0.090	0.015	0.009	5
	60% AS	0.156	0.032	0.010	8
	CTX
	0% AS	0.053	0.006	0.032	1
	60% AS	0.517	0.003	0.034	7
P167G	CAZ
	0% AS	0.130	0.030	0.026	6
	60% AS	ND	ND	ND	NDc
E166Y/P167G	CAZ
	0% AS	0.085	0.017	0.023	3
	60% AS	0.138	0.031	0.021	5
W165Y/E166Y/P167G	CAZ
	0% AS	0.130	0.020	0.031	3
	60% AS	0.670	0.090	0.037	16
W165Y/E166Y/P167G/L201P	CAZ
	0% AS	0.124	0.040	0.029	3
	60% AS	0.620	0.070	0.034	15

^a The hydrolysis rate for ceftazidime was analyzed with the integrated branched pathway equation (Equation 2). Saturating concentrations of ceftazidime (>V_max) were used (50 μm ceftazidime was used except in the case of E166Y and P167G, where 100 μm substrate was used because of their higher K_m). The data are compiled from three separate experiments with an S.D. of less than 10%. v_i is the initial velocity, v_ss is the steady-state velocity, and k is the rate constant representing the change. Presented rates are adjusted for enzyme concentration (1 μm enzyme for all mutants except 0.5 μm for W165Y/E166Y/P167G).

^b The burst amplitude is calculated as the ratio of [P]/[E] by extrapolating the progress curve at the steady-state phase to time 0.

^c ND, not determined.