pages 1-8 Received lune 15, 1998. Accepted July 6, 1998. NOVEL SUBSTRATES OF YEAST ALCOHOL DEHYDROGENASE--4. ALLYL ALCOHOL AND ETHYLENE GLYCOL Svetlana Trivid 1 and Vladimir Leskovac 2. I Faculty of Science Novi Sad and 2"Faculty of Technology Novi Sad, Bulevar Cara Lazara I, YU-21000 Novi Sad, Yugoslavia SUMMARY: In the present work, we have determined the steady-state kinetic constants for yeast alcohol dehydrogenase-catalyzed oxidation of allyl alcohol (H2C=CH.CH2OH) and ethylene glycol (HOCHg.CH2OH) with NAD at ph 8.0; also, a kinetic mechanism for the former reaction was determined at the same ph. In addition, it was found that acrolein is a potent inhibitor of yeast alcohol dehydrogenase. INTRODUCTION Yeast alcohol dehydrogenase (EC 1.t.1.1, constitutive, cytoplasmic) has a much narrower substrate specificity than equine liver enzyme (Eklund and Branden, 1987). Previously, it was reported by Bergmeyer (1970) that yeast enzyme catalyzes the reversible reduction of glycol aldehyde by NADH, forming ethylene glycol. Lutsdorf and Megnet (1968)reported that yeast enzyme catalyzes the oxidation of allyl alcohol to the toxic compound acrolein; in both cases, the steadystate kinetic constants for the enzymatic reactions were not reported. In this work, we have determined the steady-state kinetic constants for the yeast alcohol dehydrogenase-catalyzed oxidations of allyl alcohol and ethylene Copyright 9 1999 IUBMB 1 1039-9712/99 $12.00 + 0.00
glycol, at ph 8.0. Since acrolein, the product of the enzymatic oxidation of allyl alcohol, is a chemically reactive compound, we have also investigated its chemical reactions with an enzyme protein itself. MATERIALS AND METHODS Yeast alcohol dehydrogenase (lyophilized) was obtained from Boehringer. Specific activity of enzyme with ethanol was 300 U/mg of enzyme protein, estimated at ph 9, according to Bergmeyer (1970). The concentration of enzyme protein in solution was determined according to Hayes and Velick (1954), and the concentration of enzyme active sites by the fluorescent method of Leskovac et al. (1993). NAD and NADH were purchased from Sigma; allyl alcohol (99+ %) and ethylene glycol (99+ %) were purchased from Aldrich, and used without further purification. All other chemicals were of the highest grade purity, obtained from commercial sources. Absorption spectra were recorded from 230 nm - 800 nm in a spectrophotometer SPECORD UV VIS, Carl Zeiss, Jena (Germany), in thermostated cuvette holders at 25~ Concentrations of coenzymes were determined from their molar extinction coefficients at ph 7.0 (M~cm~): NAD + 18.000 at 260 nm and NADH 6200 at 340 nm (Bergmeyer, 1970). Enzyme reaction rates were determined from initial linear phase of reaction progress curves in 0.1M sodium pyrophosphate buffer ph 8.8 or in 0.1 M Tris.HCI ph 8.0, supplemented with 0.5 mm EDTA. Initial rate data were fitted with the SEQUEN and NONCOMP Fortran programs of Cleland (1979), to following equations: V1AB VJeo =... ( 1 ) KiAKB + KBA + KAB + AB 89 VJeo =... ( 2 ) KB (1 + [NADH]/Kis) + A(1 + [NADH]/K,) where Vo is the initial rate (M.s~), eo the concentration of enzyme active sites (M), V~ the maximal catalytic constant (sl), KA and KB the Michaelis constants for NAD and aldehydes (M), KiA the inhibitory constant for NAD (M), and A and B the concentration of NAD* and alcohols (M), respectively; K, and Kis (M) are inhibitory constants for NADH (Cleland, 1970).
RESULTS AND DISCUSSION Table 1 shows the steady-state kinetic constants for yeast alcohol dehydrogenase-catalyzed oxidation of ethanol, allyl alcohol and ethylene glycol. Ethylene glycol is a poor, while allyl alcohol is an excellent substrate of yeast enzyme, comparable to or even better than ethanol. TABLE 1. Steady-state kinetic constants for various alcohols at 25~ Ethanol" Allyl alcohol b Ethylene glycol Buffer 0.1 M phosphate 0.1 M Tris.HCI 0.1 M Tris.HCI ph 8.1 8.0 8.0 Vl s 4 555 546 _+ 54 7.03 + 0.44 KA mm 0.118 0.52 +0.09 0.37 +0.06 KiA mm 0.385 0.73 + 0.21 0.55 _+ 0.05 Ka mm 18.5 14.6 +3.9 444 +46 VllKA mmls 4 4703 1058 _+92 19.2 + 1.8 VIIKB mm4s 4 30 37.5 + 6.7 0.0158 + 0.0007 VlKiAIKA s -1 1811 766.5 10.4 Koq pm ~ 121 18 d a Calculated from the data of Dickinson and Monger (1973). c At ph 7.0. b Calculated from the data in Figure 1. d Calculated from the data in Figure 3A.
Figure 1 shows the double-reciprocal plot for the enzymatic oxidation of allyl alcohol with NAD and the corresponding secondary plots (Segel, 1975). 10-30 30 60 90 (ALL'fL ALE0}-IOLI HI-1..... ~..... 1!N~f EREEPTIt ~ -2 0 2 4 6 [NAI) +, mh ]-1 -? 0 "2 ~, 6 i NAD* mh] "1 FIGURE 1. Double reciprocal plot for the enzymatic oxidation of allyl alcohol with NAD In the primary plot (above), increasing concentrations of allyl alcohol were oxidized with (from top to bottom): 145, 216, 356 or 695 pm NAD in the presence of enzyme (1.9 rim), in 0.1 M Tris.HCl buffer ph 8.0, 25~
Figure 2 shows the double-reciprocal plot for the product inhibition with NADH, of allyl alcohol oxidation with NAD + (Segel, 1975). 1IV 0 1 sec x I0001 x ~/ -30 0 30 60 90 [ALLYL ALCOIt0L,M ]-1 INTERCEP I IS xio00) / - - SLOPE (,u M.s~ o/----~ 150 / ~oo p/,o 5O tnad+]= oo -I00 0 I00 ~IADII (um) I /. J -I00 I00 NABI4 {~,MI FIGURE 2. Product inibition by NADH. Primary plot (above): increasing concentrations of allyl alcohol were oxidized with 0.52 mm NAD +, in the presence of enzyme (1.2 nm) and (from top to bottom): 146, 74,45 or 0 I~M NADH, in 0.1 M Tris.HCI buffer, ph 8.0. Secondary graphs: (bottom left) I~i= 125 pm, and (bottom right) t~s = 58 p.m.
Comparison of both double reciprocal plots indicates the following properties of this enzymatic reaction, intersection points in the IV quadrant of the primary graphs in Figure 1 and in Figure 2, do not have the same coordinates. Also, intersection points in the IV quadrant of the secondary graphs in Figure 2, do not have the same coordinates. If the addition of substrates in the forward direction, oxidation of alcohol, was ordered, both intersection points should be identical. In the first case, the coordinate of the intersection point should be equal (I/V1)(1-KiA/KA) on ordinate, and in the second case equal KiQ on abscissa (Segel, 1975). A double reciprocal plot for the product inhibition of allyl alcohol oxidation with NAD shows that NADH is fully competitive with NAD at ph 8 (data not shown). This indicates that the kinetic mechanism for this reaction can not be rapid equilibrium random. Therefore, by elimination, we conclude that the order of addition of substrates on the alcohol side of reaction, is steady-state random (Segel, 1975): /\ \/ A E EAB > P, Q, E ( 3 ) B Thus, the kinetic mechanism with allyl alcohol is identical with the kinetic mechanism found previously for ethanol (Dickinson and Dickenson, 1978). The product of the enzymatic oxidation of allyl alcohol is acrolein, a chemically reactive compound; for this reason, kinetic experiments in Figures 1 and 2 were conducted in TrisHCI buffer, which can trap acrolein as a Schiff base. Figure 3A shows the determination of the equilibrium constant for the enzymatic oxidation of allyl alcohol. Figure 3B shows that, if the enzymatic reaction is conducted in a pyrophosphate buffer, the enzyme is rapidly inactivated. It is rapidly
inactivated by the product of the oxidation of allyl alcohol, but remains unaffected by acetaldehyde; thus, acrolein appears to be a potent inhibitor of yeast enzyme. -1 4- :SZ -2 100 (~B,, 80 60 --J -4 ~0-5 L I I I ~. E I 6? 8 9 0 5 10 15 ph rime (min) 20 20 FIGURE 3. (A) Determination ofkeq. Allyl alcohol (143, 48, 9.5, 1.4 or 0.5 ram) was oxidized with NAD (0.96 mm) in 0.1 M sodium phosphate or in 0.1 M sodium pyrophosphate buffers, ph 6.06, 7.07, 7.86, 8.95 or 9.55, in the presence of 83 nm enzyme. After the equilibrium was attained, K~q was calculated from the relationship: K~q/[H = [H2C=CH,CHO][NADH]/[H2C=CHCH2OH][NAD*]. (B) Inhibition of enzyme by acrolein. Allyl alcohol (270 mm) (o) or ethanol (320 mm) (A) were incubated with NAD (1 mm) and enzyme (0.17 #M) in 0.1 M sodium pyrophosphate buffer, ph 8.85 at 25~ At times indicated, small aliquots were removed and assayed for activity in 0.1 M Tris.HCI buffer ph 8.0.
The concentrations of allyl alcohol and ethylene glycol were successfully determined enzymatically by the semicarbazid method of Bergmeyer (1970), developed for the determination of ethanol. Allyl alcohol, acrolein, ethylene glycol and glycol aldehyde are valuable industrial chemicals. Therefore, a special interest deserves the enzymatic oxidation of allyl alcohol, an excellent substrate of yeast enzyme, very similar to ethanol. Also, an elegant work of Wills (1976) shows that allyl alcohol can serve as a very selective mutagen for baker's yeast. REFERENCES Bergmeyer, H. U. (1970) Methoden der enzymatischen Analyse, Verlag Chemie, Weinheim/Bergstrasse. Cleland, W. W. (1970) in The Enzymes (Boyer, P. D., Ed.) Vol. II, pp 1-65, Academic Press, New York. Cleland, W. W. (1979) Methods EnzymoL 63, 103-137. Dickinson, M. F. and Monger, G P. (1973) Biochem. J. 131,261-270. Dickinson, F. M. and Dickenson, C. J. (1978) Biochem. J. 171,629-637. Eklund, H. and Branden, C.-I. (1987) in Biological Macromolecules and Assemblies (Jurnak, F. A., McPherson, A. & Avramovi~, J., Eds.) Vol. 5, pp 75-142, Wiley Interscience, New York~ Hayes, J. H. and Velick, S. F. (1954) J. BioL Chem. 207, 225-232. Leskovac, V., Trivi(~, S. and Panteli(~, M. (1993) Anal. Biochem. 214, 431-434. Lutsdorf, U. and Megnet, R. (1968) Arch. Biochem. Biophys. 126, 933-944. Segel, I. H. (1975) Enzyme Kinetics, Academic Press, New York. Wills, C. (1976) Nature 261, 26-29.