Oxidation of methanol, ethylene glycol, and isopropanol with human alcohol
dehydrogenase family and the inhibition by ethanol and 4-methylpyrazole
Shou-Lun Leea, Hsuan-Ting Shihb, Yu-Chou Chib, Yeung-Pin Lib,c, Shih-Jiun Yinb,*
a
Department of Biological Science and Technology, China Medical University,
Taichung, Taiwan b
Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan c
Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung,
Taiwan
*Corresponding author. Tel.: +886 2 87923100x18800.
ABSTRACT
Human alcohol dehydrogenase (ADH) family comprises multiple isozymes with wide
substrate specificity and ethnic distinct allozymes. ADH catalyzes the rate-limiting
step in metabolism of various primary and secondary aliphatic alcohols. The oxidation
of common toxic alcohols, that is, methanol, ethylene glycol, and isopropanol in the
context of human ADH family remains poorly understood. Kinetic studies were
performed in 0.1 M sodium phosphate buffer, at pH 7.5 and 25 oC, containing 0.5 mM
NAD+ and varied concentrations of substrate. KM values for ethanol with recombinant
human class I ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, and ADH1C2, and
class II ADH2 and class IV ADH4 were determined to be in the range of 0.12 to 57
mM, for methanol to be 2.0 to 3,500 mM, for ethylene glycol to be 4.3 to 2,600 mM,
and for isopropanol to be 0.73 to 3,400 mM. ADH1B3 appeared to be inactive toward
ethylene glycol, and ADH2 and ADH4, inactive with methanol. The variations for
Vmax for the toxic alcohols were much less than that of the KM across ADH family.
4-methylpyrazole (4MP) was competitive inhibitor with respect to ethanol for
ADH1A, ADH1B1, ADH1B2, ADH1C1 and ADH1C2, and noncompetitive inhibitor
for ADH1B3, ADH2 and ADH4, with the slope inhibition constants (Kis) for the whole family being 0.062 to 960 µM and the intercept inhibition constants (Kii), 33 to 3,000 µM. Computer simulation studies using inhibition equations in the presence of
alternate substrate ethanol and of dead-end inhibitor 4MP with the determined
corresponding kinetic parameters for ADH family, indicate that the oxidation of the toxic alcohols up to 50 mM are largely inhibited by 20 mM ethanol or by 50 µM 4MP with noticed exceptions. The above findings provide an enzymological basis for
clinical treatment of methanol and ethylene glycol poisoning by 4MP or ethanol with
pharmacogenetic perspectives.
Keywords:
Human alcohol dehydrogenase family
Methanol
Ethylene glycol
Isopropanol
4-Methypyrazole
1. Introduction
Human alcohol dehydrogenase (ADH) constitutes a complex enzyme family that is
unique with wide variability of kinetic characteristics and allelic variations among
racial populations [1–3]. Primarily based on the homology of primary structure and
chromosomal organization of the ADH gene cluster, and also on the electrophoretic
mobility, the Michaelis constants for ethanol, the sensitivity to pyrazole inhibition and
the immunochemical features, human ADH family members have been categorized
into five classes [4,5]. The class I ADH contains multiple forms, that is, ADH1A (previously denoted αα), ADH1B (ββ), and ADH1C (γγ). The class II to IV ADHs contain a single form each, that is, ADH2 (ππ), ADH3 (χχ), and ADH4 (µµ or σσ), respectively. ADH1B and ADH1C exhibit functional polymorphisms [3,6]. Alleles
ADH1B*1 (encoding the β1 subunit polypeptide) and ADH1B*2 (encoding β2 subunit)
are predominant among Caucasians and East Asians, respectively; ADH1B*3 (encoding β3 subunit) is found exclusively in Africans and some tribes of American Indians. ADH1C*1 (encoding γ1 subunit) and ADH1C*2 (encoding γ2 subunit) are about equally distributed among Caucasians and American Indians, but the former is
highly prevalent among the East Asian and African populations. The ADH family has
been involved in the metabolism of a wide variety of physiological and
S-nitrosothiols, lipid peroxidation products, ω-hydroxyfatty acids as well as
xenobiotic primary and secondary alcohols and aldehydes [1–3,7]. Currently, class V
ADH is the only family member having no available data for catalytic function due to
its extremely labile activity [7].
Intoxications with ethylene glycol, methanol, and isopropanol are among the most
common ingestions [8]. Toxicity is related to the production of toxic metabolites by
ADH and aldehyde dehydrogenase, that is, glycolate and oxalate in ethylene glycol
poisoning and formate in methanol poisoning [9,10]. In contrast, metabolite acetone
appears less toxic than the parent compound isopropanol [8]. ADH is the rate-limiting
step in metabolism of these common industrial toxic alcohols [8–10]. Inhibition by
alternate substrate ethanol and dead-end inhibitor 4-methylpyrazole (4MP; fomepizole)
of ADH in combination with hemodialysis has been widely used for treatment of
severe ethylene glycol and methanol poisonings [8–14]. Hemodialysis is
recommended for severe isopropanol intoxication which can remove both isopropanol
and acetone effectively [8–10]. Although ethanol and 4MP are accepted antidotes,
their enzymological basis for inhibition of ethylene glycol and methanol oxidation
with individual human ADH isozymes and allozymes remains unclear. In this report,
inhibitions at pharmacologically relevant alcohol levels in the context of ADH family.
Isopropanol was also included as a comparison of secondary toxic alcohol.
2. Materials and methods
2.1. Expression and purification of human ADH family
The expression of recombinant enzyme in Escherichia coli and purification to
apparent homogeneity for human ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2,
ADH2, and ADH4 were as described previously [15,16]. Human ADH1A was
expressed and isolated essentially as those procedures for ADH2 [16] with slight
modifications in the final purification step of 5’-AMP-Sepharose affinity
chromatography, which was equilibrated with 10 mM Hepes, pH 7.0, at 4 oC instead
of the 10 mM sodium phosphate, pH 6.5 [17]. All of the isolated recombinant ADH
forms exhibited a single Coomassie blue–staining protein band with a molecular mass
of 40,000 Da on sodium dodecyl sulfate–polyacrylamide gel electrophoresis by a
PhastSystem according to the manufacturer’s protocol (Amersham Biosciences,
Bucks, UK). Protein concentration was determined by the method of Lowry et al. [18]
using bovine serum albumin as the standard.
2.2. Kinetic analysis
Kinetic studies were performed in 0.1 M sodium phosphate at pH 7.5 and 25 oC,
presence of inhibitor. It has been reported that cytosolic NAD+ concentration in rat
hepatocytes was ca. 0.5 mM [19]. The enzyme activity was determined by monitoring
the production of NADH at 340 nm using an absorption coefficient of 6.22 mM-1cm-1
or at 460 nm for emission of the fluorescence. Enzyme activity units (U) are
expressed as micromoles of NADH formed per minute. Steady-state kinetic data were
analyzed by nonlinear least-squares regression using the Cleland programs of HYPER,
COMP, NONCOMP, and UNCOMP [20]. Initial velocity data were fit with HYPER
program to the Michaelis–Menten equation.
v= Vmax × S
KM + S (1)
The data from dead-end inhibition studies were fit with one of the following linear
inhibition equations, that is, the COMP program for competitive inhibition, the
NONCOMP program for noncompetitive inhibition, or the UNCOMP program for
uncompetitive inhibition, respectively.
v= Vmax × S KM (1 + I/Kis) + S (2) v= Vmax × S KM (1 + I/Kis) + S (1 + I/Kii) (3) v= Vmax × S KM + S (1 + I/Kii) (4)
where Vmax is the maximum velocity, S is the substrate concentration, KM is the
Michaelis constant, I is the inhibitor concentration, and Kis and Kii are the slope
by evaluating the standard errors of the kinetic constants and the residual variance
[20]. In cases where the intercepts and/or slopes did not vary greatly with inhibitor
concentration, Student’s t-tests were applied to determine if they were significantly
different. All of the kinetic measurements were performed in duplicate. Values
represent means ± standard error of the mean (SEM). The coefficients of variation of
the KM and Vmax values were usually less than 15% and those of the inhibition
constants were less than 19%.
For evaluation of competitive inhibition of oxidation of toxic alcohols by ethanol, the
following equation for enzymes catalyzing two reactions simultaneously was
employed [21].
va= Va × Sa
Ka (1 + Sb/Kb) + Sa (5)
where Va is the maximum velocity of toxic alcohol, Sa and Sb are the concentrations of
toxic alcohol and ethanol, respectively; and Ka and Kb are the Michaelis constants for
toxic alcohol and ethanol, respectively.
Assuming toxic alcohol and ethanol are present in the same concentration, the activity
ratio for the toxic alcohol to the ethanol with ADH isozymes/allozymes will be
va vb =
Va/Ka
where Va and Vb are the maximum velocities, and Ka and Kb are the Michaelis
constants for toxic alcohol and ethanol, respectively.
3. Results
KM and Vmax for ethanol, methanol, ethylene glycol, and isopropanol with human ADH
family are shown in Tables 1 and 2, respectively. The KM values varied tremendously
larger than did the Vmax values for the ADH members. With respect to ethanol and
isopropanol, ADH isozymes/allozymes exhibited 2,500 and 4,700-fold variations in
KM while showed 130 and 80-fold variations in Vmax, respectively. ADH1B3 was
virtually inactive toward ethylene glycol, and both ADH2 and ADH4 were inactive
with methanol. ADH1A, ADH1B1, ADH1B2, ADH1C1, and ADH1C2 displayed
3,600, 190, 650, 4,800, and 1,100-fold variations, respectively, for KM for ethanol and
the three toxic alcohols. The corresponding catalytic efficiencies, Vmax/KM, for ADH
family are shown in Table 3. The ratios of the catalytic efficiency for toxic alcohol to
that for ethanol represent relative activity of the toxic alcohol to the ethanol with ADH,
as the two alternate substrates are present in equimolar concentrations. Methanol,
ethylene glycol, and isopropanol are tremendously less efficient than ethanol for the
ADH isozymes and allozymes studied (the relative Vmax/KM ranging from 0.0063% to
2.3%), with the only exception that ADH1A exhibited 5.9-fold greater catalytic
Inhibitions of oxidation of a wide range of methanol, ethylene glycol, and isopropanol
by the fixed alternate substrate ethanol with human ADH family are shown in Fig. 1.
Between 10 to 50 mM methanol, the inhibitions by 20 mM ethanol for ADH1B1,
ADH1C1, and ADH1C2 were calculated to be 99.4 to 97.1%, for ADH1A to be
80.9–80.7%, for ADH1B2 to be 95.1–94.5%, and for ADH1B3 to be 25.9–25.7%.
Between 10 to 50 mM ethylene glycol, the inhibitions by 20 mM ethanol for
ADH1B1, ADH1C1, and ADH1C2 were calculated to be 99.6 to 98.4%, for ADH1A
to be 80.6–79.3%, for ADH1B2 to be 95.2–94.9%, for ADH2 to be 58.3–56.1%, and
for ADH4 to be 30.2–29.9%. At 10, 20, and 50 mM isopropanol, the inhibitions by 20
mM ethanol for ADH1B1, ADH1C1, and ADH1C2 were calculated to be 98.9 to
93.7%, for ADH1A to be 22.5, 13.0, and 5.8%, respectively, for ADH1B2 to be
95.0–93.8%, for ADH1B3 to be 25.7–24.8%, for ADH2 to be 58.3–56.1%, and for
ADH4 to be 30.2–30.0%.
Steady-state kinetic studies showed that 4-methylpyrazole (4MP) was a competitive
inhibitor versus ethanol for ADH1A, ADH1B1, ADH1B2, ADH1C1, and ADH1C2,
and a mixed-type noncompetitive inhibitor against ethanol for ADH1B3, ADH2, and
ADH4. The determined corresponding inhibition constants are shown in Table 4. The
illustrates inhibitions of oxidation of a wide range of ethanol and toxic alcohols by the
fixed dead-end inhibitor 4MP. Between 20 to 50 mM ethanol, the inhibitions by 50 µM 4MP for ADH1A, ADH1B3, and ADH1C1 were calculated to be 88.8 to 65.9%, for ADH1B2 and ADH1C2 to be 72.6–50.5%, for ADH1B1 to be 9.6–4.1%, for
ADH2 to be 3.6–3.2%, and for ADH4 to be 7.9–6.1%. Between 20 to 50 mM methanol, the inhibitions by 50 µM 4MP for ADH1A, ADH1B2, ADH1C1, and ADH1C2 were calculated to be 99.9 to 97.6%, and for ADH1B1 and ADH1B3 to be 89.4–78.1%. Between 20 to 50 mM ethylene glycol, the inhibitions by 50 µM 4MP for ADH1A, ADH1B1, ADH1B2, ADH1C1, and ADH1C2 were calculated to be 99.8
to 88.0%, for ADH2 to be 4.9–4.7%, and for ADH4 to be 10.4–10.3%. Between 20 to 50 mM isopropanol, the inhibitions by 50 µM 4MP for ADH1B2, ADH1C1, and ADH1C2 were calculated to be 99.4 to 97.5%, for ADH1B1 and ADH1B3 to be
89.2–66.6%, for ADH1A to be 59.5–37.5%, for ADH2 to be 4.9–4.7%, and for ADH4
to be 10.4–10.3%.
4. Discussion
To our knowledge, in the context of human ADH family this is the first report on
kinetic properties of methanol, ethylene glycol, and isopropanol which are some of
the most common ingested toxic alcohols in comparison with that of ethanol, and on
Food and Drug Administration of the US as antidote for the toxic alcohol poisoning,
at a near physiological pH and cytosolic NAD+ concentration. We found a
considerable large variation for KM and Vmax for the toxic alcohols and also for
inhibition pattern and constants for 4MP within class I ADHs and an even larger
variation across class I, II, and IV ADHs. Most interesting is the significant variation
in KM for the toxic alcohols for class I ADH1B allozymes, suggesting that there may
be ethnic distinctions for metabolism of the toxic alcohols. It has been firmly
documented that the ADH1B*2 allele can protect against development of alcoholism
across ethnic groups [3,6].
Class I, II, and III ADHs are predominantly expressed in human liver, the major organ
for metabolism of ingested alcohols [3,22]. Class IV ADH is uniquely expressed in
stomach and upper digestive tract that it contributes to gastric first-pass metabolism of
ethanol [22,23] and toxic alcohols. Class III ADH was not included in the present
study because of its negligible role in metabolism of ethanol and the toxic alcohols. It
has been reported that human ADH3 was unsaturable with ethanol [24], inactive
toward methanol and ethylene glycol at the pH-optimum 10.0 [25], and insensitive to
The KM values for ethanol for the studied human ADH family are in following
decreasing order: ADH4 > ADH2 > ADH1A > ADH1B2 > ADH1C2 ≈ ADH1C1 >
ADH1B1, with the exception of ADH1B3 being the highest KM. X–ray
crystallographic studies indicate that class I and class IV ADH isozymes/allozymes
possess grossly similar but clearly discernible topology at the bottom of the
hydrophobic substrate binding site adjacent to the catalytic zinc ion [26–29], that can
largely explain the substrate affinity to the enzyme. Class II and IV ADHs in general
exhibit higher KM for ethylene glycol and isopropanol than do the class I enzymes
with some exceptions. ADH1A uniquely exhibits the largest KM for methanol, except
that of ADH1B3, among class I ADHs but displays the smallest KM for isopropanol
among all of the ADH family studied. This can largely be attributed to a single amino
acid substitution of Ala in ADH1A for Phe-93 in all of the remaining ADH family
members except Tyr-93 in ADH2. Smaller alanyl residue at this position allows
effective binding to more bulky secondary alcohol isopropanol and hence a much less
effective binding to the smallest substrate methanol [29,30]. Indeed, ADH1A exhibits
the highest catalytic efficiency for isopropanol compared with that for ethanol,
methanol, and ethylene glycol. Furthermore, the catalytic efficiency for isopropanol
for ADH1A is greatest among those for the class I, II, and IV ADHs studied. It has
ethanol oxidation in human ADH family [3]. For those ADH forms exhibiting
considerably lower Vmax for some of the toxic alcohols compared with that of ethanol,
it suggests a shift of rate-limiting step in catalysis, possibly to the hydride transfer.
Human class I ADH isozymes/allozymes exhibit a 95-fold variation in slope
inhibition constants (0.062–5.9 µM) for 4MP with respect to ethanol. Previous studies using a mixture of human class I isozymes isolated from the autopsy liver, reported
Kis for 4MP, 0.21 µM [31] and 0.09 µM [32]. Class II and class IV ADHs exhibit
much higher Kis for 4MP than do the class I enzymes. X–ray crystallographic and
site-directed mutagenesis studies provide evidence that Met-141 directly influences
the binding of 4MP in ADH4 [28,33]. It is interesting to note that ADH1B3, ADH2,
and ADH4 revealed a mixed-type noncompetitive inhibition of 4MP versus ethanol.
This result confirms the previous observation with ADH2 [34]. The noncompetitive
inhibition pattern suggests that 4MP may reversibly bind to both the E–NAD+ and
E–NADH binary complexes in catalytic cycle, as demonstrated by inhibition studies
with substrate analogs of malic enzyme [35]. The formation of E–NADH–inhibitor
complex prevents release of NADH, that is, the rate-limiting step, and hence giving
rise to the intercept inhibition effect. For competitive and noncompetitive inhibitions
and inhibitor in an Ordered Bi Bi mechanism. Recently formamide derivatives, potent
uncompetitive inhibitor against ethanol, has been developed to inhibit ethanol
metabolism in mice [36] and yet no available data for clinical trials.
The accepted target plasma ethanol concentration for treatment of ethylene glycol and
methanol poisoning is approximately 100 mg/dl (21.7 mM) [10]. The reported plasma
levels of ethylene glycol [11], methanol [13], and isopropanol [10] in intoxicated
patients can reach a high 50 mM. Our simulation results indicate that inhibitions of
oxidation of 50 mM toxic alcohols by 20 mM ethanol for human ADH family are
quite effective (inhibition ≥ 80%), except for methanol for ADH1B3 (25.7%) and for
ethylene glycol for ADH2 (56.1%) and ADH4 (29.9%). At therapeutically attainable plasma levels of 4MP [9], inhibition of oxidation of 50 mM toxic alcohols by 50 µM 4MP is highly effective (inhibition ≥ 88.0%), except for methanol for ADH1B1
(78.1%) and for ethylene glycol for ADH2 (4.7%) and ADH4 (10.3%). Of human
ADH family, ADH1B and ADH2 appeared to be with the highest and the second
highest protein contents in liver, respectively [17], and ADH4 is a high-activity
isozyme expressed in the stomach [23]. Therefore, the efficacy for treatment of the
toxic alcohol poisoning by ethanol or 4MP may potentially vary for patients carrying
ADH2.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
We thank Dr. Thomas D. Hurley and Dr. William F. Bosron, Department of
Biochemistry and Molecular Biology, Indiana University School of Medicine, for the
generous gift of the expression vectors for human ADH1A, ADH1B allozymes,
ADH2, and ADH4, and Dr. Jan-Olov Höög, Department of Medical Biochemistry and
Biophysics, Karolinska Institutet, Sweden, for kind providing the expression vector
for human ADH1C2. This work was supported by a grant from the National Science
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Figure Legends
Fig. 1. Inhibition of oxidation of (a) methanol, (b) ethylene glycol, and (c)
isopropanol by alternate substrate ethanol with human ADH family. Enzyme activity
ratio was simulated at 0.5 mM NAD+ and varied concentrations of toxic alcohol in the
presence (vi) to that in the absence (vo) of 20 mM ethanol using Eq. 5. For the
corresponding kinetic constants of ADH family for the simulation, see Tables 1–3.
Fig. 2. Inhibition of oxidation of (a) ethanol, (b) methanol, (c) ethylene glycol, and (d)
isopropanol by dead-end inhibitor 4-methylpyrazole with human ADH family.
Enzyme activity ratio was simulated at 0.5 mM NAD+ and varied concentrations of
alcohol in the presence (vi) to that in the absence (vo) of 0.05 mM 4MP using Eq. 2,
except using Eq. 3 for ADH1B3, ADH2 and ADH4. For the corresponding kinetic
Table 1
KM values (mM) for ethanol and toxic alcohols of human ADH family
Substrate Class I Class II Class IV
ADH1A ADH1B1 ADH1B2 ADH1B3 ADH1C1 ADH1C2 ADH2 ADH4
Ethanol 4.7 ± 0.2 0.023 ± 0.001 1.0 ± 0.1 57 ± 2 0.12 ± 0.01 0.16 ± 0.01 14 ± 1 46 ± 2 Methanol 2,600 ± 500 2.0 ± 0.2 290 ± 20 3,500 ± 300 570 ± 90 180 ± 20 –b –b Ethylene glycol 440 ± 40 4.3 ± 0.4 650 ± 40 –a 53 ± 7 49 ± 4 420 ± 20 2,600 ± 300 Isopropanol 0.73 ± 0.05 1.1 ± 0.1 160 ± 10 760 ± 70 5.3 ± 0.7 6.7 ± 0.5 420 ± 60 3,400 ± 300
Enzyme activity was determined in 0.1 M sodium phosphate at pH 7.5 and 25 oC, containing 0.5 mM NAD+ and varied concentrations of substrate. Values represent means ± SEM.
a
Not determined due to the activity too low to be precisely measured up to 2.7 M ethylene glycol. b
Table 2
Vmax values (U/mg) for ethanol and toxic alcohols of human ADH family
Substrate Class I Class II Class IV
ADH1A ADH1B1 ADH1B2 ADH1B3 ADH1C1 ADH1C2 ADH2 ADH4
Ethanol 0.37 ± 0.01 0.11 ± 0.01 4.3 ± 0.2 2.7 ± 0.1 0.65 ± 0.02 0.38 ± 0.01 0.21 ± 0.01 14 ± 1 Methanol 0.025 ± 0.001 0.033 ± 0.002 0.41 ± 0.01 0.029 ± 0.002 0.19 ± 0.02 0.080 ± 0.002 –b –b Ethylene glycol 0.17 ± 0.01 0.10 ± 0.01 1.9 ± 0.1 –a 0.27 ± 0.02 0.23 ± 0.01 0.074 ± 0.002 3.1 ± 0.2 Isopropanol 0.34 ± 0.01 0.11 ± 0.01 1.6 ± 0.1 0.35 ± 0.02 0.10 ± 0.01 0.090 ± 0.002 0.045 ± 0.002 3.7 ± 0.3 For assay conditions, see Table 1.
a
Not determined due to the activity too low to be precisely measured up to 2.7 M ethylene glycol. b
Table 3
Vmax/KM values (mU/(mg mM)) for ethanol and toxic alcohols of human ADH family
Substrate Class I Class II Class IV
ADH1A ADH1B1 ADH1B2 ADH1B3 ADH1C1 ADH1C2 ADH2 ADH4
Ethanol 79 ± 2 4,700 ± 200 4,200 ± 200 48 ± 1 5,500 ± 500 2,300 ± 100 15 ± 1 310 ± 10 Methanol 0.0098 ± 0.0008 17 ± 1 1.4 ± 0.1 0.0085 ± 0.0003 0.35 ± 0.03 0.43 ± 0.03 –b –b (0.012%) (0.35%) (0.034%) (0.018%) (0.0063%) (0.018%) Ethylene glycol 0.38 ± 0.03 24 ± 2 3.0 ± 0.1 –a 5.0 ± 0.3 4.8 ± 0.3 0.18 ± 0.01 1.2 ± 0.1 (0.47%) (0.51%) (0.072%) (0.090%) (0.20%) (1.2%) (0.39%) Isopropanol 460 ± 20 110 ± 10 10 ± 1 0.48 ± 0.03 20 ± 2 14 ± 1 0.11 ± 0.01 1.1 ± 0.1 (590%) (2.3%) (0.25%) (0.99%) (0.36%) (0.58%) (0.72%) (0.36%)
For assay conditions, see Table 1. The ratios of Vmax/KM for toxic alcohol to that for ethanol are shown in parentheses. Values of the ratios
represent relative activity of toxic alcohol to ethanol assuming the two alternate substrates are present in equimolar concentrations (refer to Eq. 6). Note that milliunits (mU) are used to express the enzyme activity.
a
Not determined due to the activity too low to be precisely measured up to 2.7 M ethylene glycol. b
Table 4
Inhibition constants of 4-methylpyrazole with respect to ethanol in human ADH family
Class Isozyme/allozyme Inhibition pattern Inhibition constant
Kis (µM) Kii (µM) I ADH1A Comp 1.2 ± 0.1 ADH1B1 Comp 0.54 ± 0.06 ADH1B2 Comp 0.96 ± 0.08 ADH1B3 Noncomp 5.9 ± 0.5 33 ± 6 ADH1C1 Comp 0.062 ± 0.005 ADH1C2 Comp 0.15 ± 0.01 II ADH2 Noncomp 960 ± 140 1,800 ± 200 IV ADH4 Noncomp 430 ± 30 3,000 ± 600
Enzyme activity was determined in 0.1 M sodium phosphate at pH 7.5 and 25 oC, containing 0.5 mM NAD+ and varied concentrations of both substrate and inhibitor. Values represent means ± SEM.
