Substrate Specificity of Alcohol Dehydrogenase

Transcription

1 0 Substrate Specificity of Alcohol Dehydrogenase Roshan Roshan Chikarmane and Jonathan White Department of Chemistry University of Oregon Eugene, OR April 26, 2014 Abstract: The substrate specificity of NAD-dependent alcohol dehydrogenase (ADH) in regards to the molecular shape of various alcohols have been determined using a unique assay system that allows the catalyzed rate of alcohol redox to be measured by visible spectroscopy. The saturation point and optimal ph environment of ADH were determined by visible spectroscopy. A similar analytical method was used on ADH-catalyzed redox reactions of saturated, ph optimized methanol, ethanol, 1-propanol, butanol, and 2- propanol solutions. Further investigation indicated that the substrate binding channel of ADH most favorably accommodated the molecular shape of ethanol, followed by 1-propanol, butanol, 2-propanol, and least favorably accommodated methanol.

2 1 Substrate Specificity of Alcohol Dehydrogenase Roshan Chikarmane and Jonathan White Introduction: Alcohol dehydrogenase (ADH) is an enzyme that catalyzes the oxidation of ethanol (EtOH) to acetaldehyde, as shown in Equation 1. Nicotinamide adenine dinucleotide (NAD) takes up two electrons (1) and one proton from the catalyzed reduction of ethanol to form reduced NADH molecule 1. The structure of the β 3β 3 human ADH isoenzyme complexed with NAD has been well characterized by X-ray crystallography as a dimeric protein, as shown in Figure 1. Each monomer is Figure 1. The quaternary structure of dimeric β 3β 3 human ADH isoenzyme complexed with NAD (purple). Individual subunits are represented in red and blue (1). composed of 373 and 393 amino acids and contains two tetracoordinate zinc cations, one for catalytic substrate binding and another to stabilize protein folding, as shown in Figure 2. The catalytic zinc ligates

3 2 Figure 2. Tertiary structure of β 3β 3 human ADH isoenzyme complexed with NAD (purple), a neighboring catalytic Zn 2+ (yellow), and a structural Zn 2+. Composed of 11 α-helices (blue) and 18 β-sheets (red) (1). with amino acids cysteine-46, cysteine-174, histidine-67, and the hydroxyl oxygen of the alcohol substrate, in close proximity with a NAD cofactor and is illustrated in Figure 3. The goal of this research project was to investigate how the rate of catalyzed alcohol oxidation was effected by ethanol concentration, alcohol structure (methanol, 1-propanol, 1-butanol, 2-propanol), and ph. The utilized assay system allowed the rate of reaction to be observed by the decrease in absorption at 634 nm by visible spectroscopy. NADH, produced by the ADH-catalyzed oxidation of ethanol, reduces the oxidized form of phenazine methosulphate (PMS ox) to its reduced form (PMS red). PMS red reduces blue 2,6-dichlorophenolindophenol (DCIP ox) to its colorless oxidized form (DCIP red). The assay pathway is shown in Equation 2 (3). (2)

4 3 Figure 3. Active site of β 3β 3 human ADH isoenzyme complexed with PAD (red molecule), a NAD analogue. The zinc cation (yellow) is ligated with cysteine-46, cysteine-174, histidine-67 (blue), and ethanol (red-oxygen, black-carbon, white-hydrogen) (2). Materials and Methods: A 634 Visible Spectroscopy: The absorption at 634 nm (A 634) was measured once every 15 seconds for three minutes by visible spectroscopy, immediately after 0.5 ml of ADH (stored at 0 C) was added and the solution was briefly agitated (about 0.5 seconds). Effect of substrate concentration on the rate of ADH-catalyzed alcohol oxidation A g sample of tris(hydroxymethy)aminomethane (TRIS) was dissolved in 85 ml of H 2O. The TRIS buffer ph was lowered to 8.05 with concentrated (6.0 M) HCl and diluted to 100 ml with H 2O. Three empty cuvettes were filled with 1.00 ml of 0.75 mm NAD, 0.5 ml TRIS buffer, and 1.00 ml of 1.2 mm EtOH. The A 634 Visible Spectroscopy procedure was followed with two samples, but with H 2O substituted for ADH in the

5 4 third sample. The volumes reported in Table I in addition to 1.0 ml of 0.75 mm NAD and 0.5 ml TRIS buffer were added to nine empty cuvettes. The A 634 Visible Spectroscopy procedure was followed with each of the nine samples. Table 1. Sample composition for the concentration effect of EtOH Sample # [EtOH] stock a V EtOH added b V water added b a All concentrations in mol/l; b All volumes in ml Effect of ph on rate of ADH-catalyzed alcohol oxidation Three cuvettes were filled with ml of 0.75 mm NAD, ml of 0.90 M EtOH, and 0.50 ml of M NaCH 3COO buffer (ph = 6.0) The same procedure was applied to applied to two sets of three empty cuvettes, but M TRIS buffer (ph = 9.0) and M glycine (ph = 10.0) were added to each respective set instead of M NaCH 3COO buffer (ph = 6.0). The A 634 Visible Spectroscopy procedure was followed with each of the six samples. Effect of substrate structure on rate of ADH-catalyzed alcohol oxidation Three empty cuvettes were filled with ml of 0.75 mm NAD, ml TRIS buffer, and ml of 0.90 M methanol. The same procedure was applied to three sets of three empty cuvettes, but 1-propanol, 1-butanol, and 2- propanol was added to each respective set instead of methanol. The A 634 Visible Spectroscopy procedure was followed with each of the 12 samples.

6 V max (min -1 ) 5 Results: The goal of this research project is to investigate how the rate of catalyzed alcohol oxidation effects ethanol concentration, alcohol structure (methanol, 1-propanol, 1-butanol, 2-propanol), and ph. (a) Effect of substrate concentration on the rate of ADH-catalyzed alcohol oxidation. The A 634 of solutions with varying ethanol substrate concentrations were recorded against time (minutes) since initiation of the reaction and is shown in Table 2. The absolute approximate slope of A 634 as a function of time, for each EtOH concentration ([EtOH] 0) was found to be the maximum rate (min -1 ) of reaction and is summarized in Table 2. The maximum rate of reaction (V max) is plotted against [EtOH] 0 and is shown in Figure 4. The initially positive, linear and subsequent asymptotic approach to a fixed theoretical maximum V max (as the [EtOH] 0 increases) suggests that the rate law is initially first order, but transitions to zero order. A zero order rate law with excess EtOH suggests that ADH is saturated with substrate, which precludes a further increase in V max. The saturation concentration for a 40.0% (v/v) ADH stock to remaining solution volume is 0.30 M alcohol. Table 2. Initial Ethanol Concentration vs. Maximum Rate of Reaction [EtOH]0 (mol/l) Vmax (min -1 ) Plotted in Figure [EtOH] 0 (mol/l) Figure 4. Plot of V max vs. [EtOH] 0 (mol/l) demonstrates initial first order reaction followed by zero order reaction (asymptote nearing theoretical maximum rate) as ADH approaches saturation.

7 6 (b) Effect of ph on rate of ADH-catalyzed alcohol oxidation. The A 634 of solutions with equal amounts of varying buffer ph were recorded against time (minutes) since initiation of the reaction. The absolute approximate slope of A 634 as a function of time, for each solution was found to be the maximum rate (min -1 ) of reaction and is summarized in Table 3. The maximum rate of reaction (V max) is plotted against buffer ph and is shown in Figure 5. The V max of solution containing TRIS buffer (ph = 8.00) is 4.33 times greater than the solution containing NaCH 3COO buffer (ph = 6.00) and 5.50 times greater than the solution of glycine buffer (ph = 10.0). This suggests that a TRIS buffer (ph = 8.00) provides the most favorable buffer system for the ADH-catalyzed oxidation of alcohol out of all the tested buffers. Table 3. Buffer ph vs. Vmax Buffer ph Vmax Plotted in Figure 5. V max Buffer ph Figure 5. Plot of V max vs. buffer ph demonstrates that the TRIS buffer (ph = 9.00) maintains the most favorable buffer system for the ADH-catalyzed oxidation of alcohol. (c) Effect of substrate structure on rate of ADH-catalyzed alcohol oxidation. The A 634 of solutions with varying substrate molecular structure were recorded against time (minutes) since initiation of the reaction. The absolute approximate slope of A 634 as a function of time, for each solution was found to be the maximum rate (min -1 ) of reaction, and is summarized in Table 4. The maximum rate of reaction (V max) is plotted against substrate structure and is illustrated in Figure 6. The V max for ADH-catalyzed alcohol oxidation is highest for ethanol substrate, followed by 1-propanol, butanol, 2-propanol, and methanol. Alcohol Vmax to V max for methanol oxidation (V max(oh):v max(meoh)) is 117:1 for ethanol

8 Initial Rate (min -1 ) 7 oxidation, 23.8:1 for 1-propanol, 5.94:1 for butanol, and 1.99:1 for 2-propanol. The variance in V max(oh):v max(meoh) values demonstrate a difference in substrate specificity among tested alcohols that favors the molecular shape of ethanol more than that of 1-propanol, butanol, 2-propanol, and methanol. Table 4. Substrate structure vs. Vmax Substrate Vmax Methanol Ethanol Propanol Butanol Propanol Plotted in Figure 6. 0 Alcohol Structure Figure 6. Plot of V max vs. substrate structure (ordered by size) demonstrates that oxidation of ethanol is most effectively catalyzed by ADH, followed by 1- propanol, butanol, 2-propanol, and methanol. Discussion: The hydrophobic active site of ADH is centered on a tetracoordinate zinc cation that ligates with amino acids cysteine-46, cysteine-174, histidine-67, and the hydroxyl oxygen of an alcohol substrate in close proximity with NAD, as illustrated in Figure 7. The substrate channel of NAD-dependent ADH binds more tightly to the molecular shape of ethanol than that of 1-propanol, butanol, 2-propanol, and methanol. Methanol is the least bulky molecule followed by ethanol, 1-propanol, butanol, and the most bulky 2-propanol. The trend in relative sizes versus the V max values suggests that an increase in alcohol substrate size larger than that of ethanol correlates with a decrease in binding character due to an increasing inability to sit in the active site of ADH. Such an inability would decrease the catalytic effect

9 8 of the zinc cation and account for the measured decrease in V max. The small size of methanol relative to ethanol may cause resonance within the active site that results it a semi-stable position far from the catalytic zinc, which would account for its miniscule relative V max. Further experimentation is required to identify the manner by which relative size vs. V max trend occurs. PAD Ethanol Cys-46 Zn His-67 Cys-174 Figure 7. Active site of β 3β 3 human ADH isoenzyme complexed with PAD (red molecule), a NAD analogue. The zinc cation (yellow) is ligated with cysteine-46, cysteine-174, histidine-67 (blue), and ethanol (red-oxygen, black-carbon, white-hydrogen) 2. References: (1) Davis, Gerard J.; et al. J. Biol. Chem. 1996, 271, (2) Li, Hong; et al. Biochem. 1994, 33, (3) DiJiacomo, Christopher; Krill, Allison, Vitz, Ed; J. Chem. Educ. 2005, 82,