Table 4.

pH Dependence of kinetic constants for forward and reverse reactions of H44R/H48Q and wild-type yeast ADHs^a.

	H44R/H48Q		Wild-typeb
Kinetic constant	pK	Limiting values or (at pH 7)	pK or slope	Limiting values or (at pH 7)
NAD+ and ethanol
$V_1/E_{\text{t}}$ (s−1)	pK1 7.0 ± 0.2 pK2 9.2 ± 0.3	$k_{\text{a}}$ 32 ± 9 $k_{\text{b}}$ 230 ± 50	7.3 ± 0.1	$k_{\text{a}}$ 190 ± 14 $k_{\text{b}}$ 530 ± 16
$V_1/E_{\text{t}}{K}_{\text{b}}$ (mM−1s−1)	8.5 ± 0.1	$k_{\text{b}}$ 12 ± 2	7.8 ± 0.2	$k_{\text{a}}$ 3.5 ± 0.9 $k_{\text{b}}$ 41 ± 5
$K_{\text{ia}}$ (mM)	~pH independent	0.16 (at pH 7)	8.1 ± 0.2	$k_{\text{a}}$ 0.54 ± 0.06 $k_{\text{b}}$ 4.9 ± 0.9
V1/EtKa (mM−1s−1)	8.03 ± 0.04	$k_{\text{b}}$ 1200 ± 80	−0.11 ± 0.02	1570 (at pH 7)
NADH and acetaldehyde
$V_2/{\mathrm{E}}_{\mathrm{t}}$ (s−1)	9.6 ± 0.1	$k_{\text{a}}$ 850 ± 60	7.8 ± 0.2	$k_{\text{a}}$ 3600 ± 300 $k_{\text{b}}$ 1200 ± 200
V1/EtKp (mM−1s−1)	9.5 ± 0.1	$k_{\text{a}}$ 32 ± 3	7.7 ± 0.1	$k_{\text{a}}$ 3200 ± 200
$K_{\text{iq}}$ (μM)	8.9 ± 0.2	$k_{\text{a}}$ 7.9 ± 1.4	7.4 ± 0.2	$k_{\text{a}}$ 15 ± 2 $k_{\text{b}}$ 410 ± 80
$V_2/{\mathrm{E}}_{\mathrm{t}}{k}_{\text{q}}$ (μM−1s−1)	8.8 ± 0.1	$k_{\text{a}}$ 16 ± 2	7.8 ± 0.2	32 ± 3

^aThe data points for H44R/H48Q ADH are shown in Fig. 3 with the lines calculated from the fits of the data to the logarithmic forms of the equations derived for a simple pH dependence where two forms of enzyme, E and EH, are related by the pK value, and $k_{\text{a}}$ represents the rate or dissociation ($K_{\text{ia}}$ or $K_{\text{iq}}$) constants for reaction of EH, and $k_{\text{b}}$ represents the constants for reaction of E. If only one form of enzyme is reacting, the equations are $k_{\text{obs}}=k_{\text{a}}/\left(1+K/\left[{\text{H}}^{+}\right]\right)$ or $k_{\text{obs}}=k_{\text{b}}/\left(1+\left[{\text{H}}^{+}\right]/K\right)$, and if both forms react, the “WAVE” equation applies: $k_{\text{abs}}=\left(k_{\text{a}}+k_{\text{b}}K/\left[{\text{H}}^{+}\right]\right)/(1\left.+K/\left[{\text{H}}^{+}\right]\right)$. $V_1/E_{\text{t}}$ data for H44R/H48Q ADH were fitted to $k_{\text{obs}}=\left(k_{\text{a}}+k_{\text{b}}{\text{K}}_1/\left[{\text{H}}^{+}\right]\right)/(1+{\text{K}}_1/{\text{H}}^{+}]+\left[{\text{H}}^{+}\right]/{\text{K}}_2)$, where $k_{\text{a}}$ and $k_{\text{b}}$ are the activities of monoprotonated and unprotonated forms of enzyme, and pK₁ describes the deprotonation of H₂E, and pK₂ is for deprotonation of HE. See also Refs. [6, 7]. A nonlinear least squares program was used for fitting of the logarithmic forms of the equations. Fits to more complicated mechanisms with four or five parameters were also tested, but the simplest mechanism with low standard errors (e.g., pK ± < 0.2) was considered to be acceptable and informative.

^bpH dependence graphs and data for wild-type enzyme were reported previously [6, 7, 9, 14]. Data for H44R ADH are given in [6] and data for H48Q ADH in [9].