Table 1.

Parameters for DegS Cleavage and/or OMP Peptide Binding Activity

		Activation Parameters
Enzyme	OMP Peptide	Maximum Activity (M⁻¹s⁻¹)	K_act (μM)	Hill Constant
Wild-type	none	2.9 ± 0.5	n.a.	n.a.
Wild-type	DNRDGNVYYF	730 ± 80	3.9 ± 0.8	1.8 ± 0.2
Wild-type	DNRDGNVYQF	590 ± 70	50 ± 5	1.6 ± 0.1
Wild-type	EDGEDGDYYF	290 ± 60	73 ± 10	1.3 ± 0.1
Wild-type	KRRKGKVYYF	70 ± 7	≤%1 μM^a	~1.2^a
Wild-type	YYF	2500 ± 550	29 ± 3	1.7 ± 0.1
Wild-type	YQF	2100 ± 200	260 ± 10	1.6 ± 0.1
K243D	None	210 ± 30	n.a.	n.a.
K243D	DNRDGNVYYF	1700 ± 150	0.3 ± 0.1	^b
K243D	DNRDGNVYQF	1800 ± 200	5.3 ± 1.2	1.3 ± 0.1
K243D	YYF	3400 ± 550	6.4 ± 1.6	1.4 ± 0.2
D320A	None	100 ± 20	n.a.	n.a.
D320A	DNRDGNVYYF	2100 ± 200	0.2 ± 0.1	^b
D320A	DNRDGNVYQF	2300 ± 200	6.3 ± 1.3	1.2 ± 0.1
D320A	EDGEDGDYYF	1020 ± 150	14 ± 2	1.3 ± 0.1
D320A	KRRKGKVYYF	810 ± 80^c	n.d.	n.d.
D320A	YYF	3600 ± 450	4.8 ± 1.2	1.4 ± 0.1
M319A	None	4.5 ± 1.0	n.a.	n.a.
M319A	DNRDGNVYYF	20 ± 2^c	n.d.	n.d.
M319A	DNRDGNVYQF	18 ± 2^c	n.d.	n.d.
M319A	KRRKGKVYYF	6 ± 1^c	n.d.	n.d.
M319A	YYF	120 ± 20	3.2 ± 0.4	1.1 ± 0.1
K243D/M319A	None	490 ± 50	n.a.	n.a.
K243D/M319A	DNRDGNVYYF	1100 ± 130^c	n.d	n.d
K243D/M319A	YYF	2700 ± 400	0.7 ± 0.2	1.2 ± 0.1

		Michaelis-Menten Parameters
Enzyme	OMP Peptide	V_max (s⁻¹enz⁻¹)	K_M (μM)	Hill Constant
Wild-type	DNRDGNVYYF	1.1 ± 0.2	750 ± 120	1.6 ± 0.2
Wild-type	YYF	2.6 ± 0.2	370 ± 40	1.4 ± 0.2
K243D	DNRDGNVYYF	1.8 ± 0.2	580 ± 30	1.3 ± 0.1
K243D	YYF	2.3 ± 0.1	440 ± 30	1.3 ± 0.1
D320A	DNRDGNVYYF	2.5 ± 0.3	520 ± 90	1.1 ± 0.2
D320A	YYF	3.1 ± 0.3	430 ± 50	1.2 ± 0.1
K243D/D320A	DNRDGNVYYF	2.0 ± 0.3	630 ± 100	1.1 ± 0.1
K243D/M319A	DNRDGNVYYF	1.4 ± 0.1	840 ± 90	1.2 ± 0.1
K243D/M319A	YYF	2.1 ± 0.2	490 ± 40	1.2 ± 0.1

	OMP Peptide Binding
Enzyme	K_D (μM)
Wild-type	4.6 ± 0.3
M319A	0.14 ± 0.03
K243D/M319A	0.18 ± 0.02

		Fitted MWC Allosteric Parameters
Enzyme	OMP Peptide	k_r (s⁻¹)	L₀	K_RS (μM)	K_TS (μM)	K_RP(μM)	K_TP (μM)	K_TP/K_RP	L₃ Peptide
Wild-type	DNRDGNVYYF	3.3	15000	150	505	0.20	1.36	6.8	50
Wild-type	KRRKGKVYYF	3.3	15000	150	505	0.23	0.76	3.3	420
Wild-type	DNRDGNVYQF	3.3	15000	150	505	2.1	12.8	6.1	61
Wild-type	YYF	3.3	15000	150	505	1.51	18.6	12.3	8
K243D	DNRDGNVYYF	3.3	180	150	505	0.07	0.14	2.0	21
K243D	DNRDGNVYQF	3.3	180	150	505	1.64	3.19	1.9	25
K243D	YYF	3.3	180	150	505	1.24	3.76	3.0	6.5
D320A	DNRDGNVYYF	3.3	350	150	505	0.06	0.2	3.3	11
D320A	EDGEDGDYYF	3.3	350	150	505	4.02	8.5	2.1	37
D320A	DNRDGNVYQF	3.3	350	150	505	1.09	2.98	2.8	16.4
D320A	YYF	3.3	350	150	505	0.9	3.7	4.1	5
M319A	YYF	3.3	5500	150	505	0.34	0.91	2.7	290
K243D/M319A	YYF	3.3	90	150	505	0.33	0.68	2.1	10.3

The fitted K_act was too close to the enzyme concentration (0.5 μM monomer) to determine reliable “free” peptide, and thus a Hill constant.

Binding was too tight to determine a reliable Hill constant.

Complete titration curves were not determined, but near saturation was confirmed by testing at least two peptide concentrations that differed by a 2-fold minimum.

Activation parameters were determined by experiments like those shown in Figures 2A and 2B. Values in italics are from Sohn et al. (2007); n.d., not determined; n.a., not applicable. In “OMP Peptide Binding,” the binding affinities are for the peptide fluoresceine-β-alanine-KKDNRDGNYYF. Experimental values are an average of two or more independent determinations. Errors were calculated as $\sqrt{1 / (n - 1) \sum_{1}^{n} {(value - mean)}^{2}}$ where n is the number of independent trials. In “Fitted MWC Allosteric Parameters,” L₃ is the equilibrium constant relating the peptide-saturated tense and relaxed states (see Figure 5).