Table 1.

Binding constants obtained by steady state and single turnover methodologies. Single turnover data are from experiments presented here; Km and enzymatic efficiency data are from Mundle et al.(35). Km for an enzyme such as AP endo that follows Briggs-Haldane kinetics (2) is described by the equation Km = (k_cat + k₋₁)/k₊₁. When k_cat < k₋₁, K_d is best approximated by the Km obtained by steady state measurements. In these cases [ES]_eq measured by single turnover is an underestimate, which results in an overestimate of K_d. Hence, the Km determined by steady state kinetics is the better estimator of K_d. (See text.)

Mutant	Abasic Site	Km(1) (nM)	kcat(1) (s−1)	Enzyme Efficiency(1) (M−1s−1)	Kd(2) (nM)
Wild Type	+	100	10	1 × 108	0.8(1)
Y269A	+	165	1.3	8 × 106	7±0.5
Y128A	+	140	0.5	3 × 106	52±22
Y171F	+	255	9 × 10−4	4 × 103	255
Y171H	+	>2000	4 × 10−3	2 × 103	>2000
Y171A	+	360	8 × 10−4	2 × 103	360

⁽¹⁾Data from Mundle et al.(35).

⁽²⁾K_d values derived from single turnover (3 or 4 independent experiments for each mutant) or steady state experiments⁽¹⁾, as appropriate (see text).