Table 1.

Kinetic Rate Constant Estimates determined by Progress Curve Analysis ^a

Inhibitor	k1 (M−1s−1)	SD	k3 (s−1)	SD	k4 (M−1s−1)	SD	k5 (s−1)	SD	Ki (nM)	SD
EapH1 WTb	6.0×105	2.2×105	7.4	0.8	2.0×106	1.4×105	4.3×10−5	1.3×10−5	0.021	0.005
EapH2 WT	3.1×105	4.5×104	7.8	0.2	2.9×105	4.3×104	5.9×10−4	2.1×10−5	2.11	0.32
H1 R89Mb,c	-	-	7.0	0.1	-		-		321	12
H2 R90M	2.4×105	1.3×105	7.2	1.7	2.8×105	1.3×105	5.5×10−4	4.1×10−5	2.3	1
H1 L59Fc	-	-	6.3	0.3	-		-		200	5
H2 K120M	3.2×105	5.5×104	8.3	1.1	2.6×105	1.1×105	8.3×10−4	1.8×10−5	3.8	2.0
H2 K121M	4.3×105	2.3×105	7.0	0.7	3.3×105	6.6×104	9.1×10−4	6.9×10−5	2.9	0.7
H2 D122N	3.5×105	4.6×104	7.9	1.5	2.6×105	5.8×104	3.7×10−4	1.6×10−5	1.5	0.4
H2 D122A	6.9×105	5.0×105	8.3	2.3	1.2×105	1.5×104	3.1×10−3	1.2×10−4	26	3
H2 S123A	1.9×105	5.4×104	6.6	0.5	1.6×105	7.8×104	4.1×10−4	2.6×10−5	3.2	2.1
H2 Y124F	5.5×105	1.3×105	8.4	0.2	1.2×105	3.8×104	2.2×10−3	5.1×10−5	18.6	4.9
H2 T125Ac	-	-	6.2	0.2	-		-		53	4
H1 N126A	2.4×105	6.4×104	6.3	0.5	9.5×105	5.0×105	1.0×10−4	2.5×10−5	0.166	0.163
H2 N127Ac	-	-	5.8	0.5	-		-		397	66
(-)b,d	4.0×105	2.2×105	7.4	0.8	-		-		-

^aMicroscopic rate constant estimates are the average of at least three individual sets of 57 progress curves (three [elastase], three [substrate] per [elastase], and five [inhibitor] per [substrate].

^bvalues previously determined by Herdendorf and Geisbrecht.³

^cDue to the loss of the time-dependent inhibition characteristic, these mutants were analyzed by a classical 5×5 inhibition assay. The data (n = 3) were globally fit to a competitive inhibition model using SigmaPlot 10.0/Enzyme Kinetics Module 1.3 (Systat Software, Inc). The K_i estimates reported are an average of three individual data sets. Errors reported are the standard deviation. k₄ and k₅ were estimated by following the EapH2 mutants binding to immobilized elastase using surface plasmon resonance (See Table 2).

^dMicroscopic rate constant estimates are the average of 10 individual sets of 12 progress curves (two [elastase], six [substrate] per [elastase].