Table 1. Acidity Constants for Ionization of the EH·I^3– Inhibitor Complex and Dissociation Constants (K_i)_E and (K_i)_EH for Release of PGA Trianion (Scheme 3) Determined for Wildtype and Mutant Forms of TIM for Reactions at 25 °C and I = 0.1 (NaCl).

Enzyme	(Ki)E (M)a	KEHI (M)b	(Ki)EH (M)c	(Ki)E·KEHI (M–2)d	(kcat/Km) (M–1 s–1)e
cWT	≈2 × 10–2	2.9 × 10–11	5 × 10–9	5.8 × 10–13	1.1 × 107g
		(pKa ≈ 10.5)
TbbWTf	≈2 × 10–2	1.2 × 10–11	2 × 10–9	2.4 × 10–13	8.4 × 106
		(pKa ≈ 10.9)
yWT	≈2 × 10–2	4.3 × 10–11	7 × 10–9	8.5 × 10–13	8.9 × 106h
		(pKa ≈ 10.4)
yS211G	n.d	n.d		4.9 × 10–12	4.2 × 106h
yY208T	n.d	n.d		1.3 × 10–11	1.0 × 106h
yY208S	n.d	n.d		1.6 × 10–10	2.2 × 105h
yY208A	n.d	n.d		1.6 × 10–10	1.8 × 105h
yY208T/S211G	n.d	n.d		1.6 × 10–11	7.3 × 105h
TbbP166A	9.6 × 10–3	4.8 × 10–10		4.6 × 10–12	2.5 × 105g
		(pKa = 9.5)
yS211A	2.4 × 10–2	6.1 × 10–9		1.5 × 10–10	2.2 × 105h
		(pKa = 8.2)
TbbI170Af	7.1 × 10–3	1.9 × 10–8		1.3 × 10–10	8.0 × 104f
		(pKa = 7.7)
cL7R	2.6 × 10–3	1.8 × 10–7		4.7 × 10–10	5.7 × 104g
		(pKa = 6.7)
yY208F	1.6 × 10–2	4.9 × 10–7		7.8 × 10–9	9.6 × 103h
		(pKa = 6.3)

^aDetermined from the fit of the data from Figure 2 to eq 7, in cases where there is an upward break in the pH profiles from Figure 2. The value of (K_i)_E ≈ 0.02 M for wildtype TIMs was estimated as described in the text.

^bDetermined from the fit of the experimental data to eq 7, when there is an upward break in the pH profiles from Figure 2. The values for wildtype TIM were estimated as described in the text.

^cCalculated from eq 9 using values of (K_i)_EK_EHI determined from nonlinear least-squares fits of the inhibition data 7 and K_EH = 10^–3.9_.²⁰

^dDetermined from the nonlinear least-squares fit of data from Figure 2 to eq 7 or to 8, depending on whether there is an upward break in the pH profile at high pH.

^eSecond-order rate constant for TIM-catalyzed isomerization of GAP, calculated for the total concentration of the hydrated and carbonyl forms of substrate.

^fRef (14).

^gRef (34).

^hRef (35).