KINETICS AND MECHANISM OF METHANOL AND FORMALDEHYDE INTERCONVERSION AND FORMALDEHYDE OXIDATION CATALYZED BY LIVER ALCOHOL DEHYDROGENASE
Y. Pocker and Hong Li Department of Chemistry University of Washington Seattle, WA 98195
INTRODUCTION Liver alcohol dehydrogenase (LADH) is a well-characterized protein. Both its primary and tertiary structures are known, as are some of the catalytic mechanisms, isozyme differences, evolutionary divergences and a number of enzymatic properties (J6rnvall, 1970; Branden, et aI., 1975; Klinman, 1981; Eklund and Branden, 1983; Pettersson, 1987; Pocker, 1989). In contrast, relatively little is known about its interaction with methanol, although there is extensive structural homology between this compound and ethanol. Compared with ethanol, methanol is an agent with a much more pronounced toxicity, notably, the ability to cause visual impairment (Jacobsen, et al., 1986). Moreover, there is increasing concern about the potential of formaldehyde, a metabolite of methanol, to act as a carcinogen (Clary, et al., 1983). In regard to its oxidation by LADH, methanol also shows a much slower reaction rate. There are significant differences in enzymatic behavior between methanol and other primary alcohols. Studies on the interaction of methanol with LADH will enable us to understand the biological behavior as well as the mechanism of methanol metabolism. Due to its relatively slow reaction rate as compared to ethanol, methanol was generally believed not to be a substrate of liver alcohol dehydrogenase (Winer, 1958; Wratten and Cleland, 1965). Later studies have shown that it is a substrate of alcohol dehydrogenases from both liver and yeast (Mani, et aI., 1970; Pocker, et aI., 1987a & 1987b). However, prior to the current study, no detailed rationale has been put forward for the enzymatic oxidation of methanol. The oxidation mechanism of ethanol and many other primary alcohols by LADH has been established to be an ordered bi-bi process (the upper pathway in Scheme I). That is to say, the binding of NAD+ precedes the binding of the alcohol with the ratelimiting step being the dissociation of NADH from the enzyme-NADH complex (Wratten and Cleland, 1963). For the oxidation of methanol, however, transient and
Enzymology and Molecular Biology of Carbonyl Metabolism 3 Edited by H. Weiner et al., Plenum Press, New York, 1990
315
steady-state kinetic studies with deuterated analogues have shown that the rate-limiting step is the hydride transfer in the enzyme-NAD+ -methanol complex (Brooks and Shore, 1971). A (EA)
Kia
P
B
Kp
Kb (EAB)
k
(EPQ)
r----E (EP)
(EB)
Ka B
Kiq
(EQ)
E
Kib
Q
A
Kip
Kq Q
P
SCHEME I. Random Mechanism for Liver Alcohol Dehydrogenase. E: LADH; A: NAD+; B: alcohol; P: aldehyde; Q: NADH.
The purpose of this study was to delineate, in detail, the kinetics and mechanism of the reactions of methanol and its metabolites with liver alcohol dehydrogenase. Our results indicate that methanol binds to a similar site in the enzyme as does ethanol, but due to the lack of an appreciable hydrophobic chain, it exhibits different kinetics and is the only simple primary alcohol found to follow a rapid equilibrium random mechanism (combined upper and lower pathways in Scheme I). EXPE~ENTALPROCEDURES
Horse LADH, NAD+, and NADH were obtained from Sigma Chemical Company. Pyrazole (recrystallized twice before use) was obtained from Aldrich Chemical Company. All other chemicals used were reagent grade. Methanol solutions were obtained by dissolving a weighed amount of absolute methanol in buffer. Formaldehyde solutions were prepared by distilling an aqueous solution of methenamine acidified with an equivalent amount of H2S04. The resulting distillate was neutralized, redistilled, and its concentration checked both gravimetrically with dimethylcyclohexanedione (Weinberger, 1931), and enzymatically using an excess of NADH in the presence of LADH. LADH, NAD+ and NADH solutions were prepared immediately before each experiment. The LADH solution was kept at OOC to prevent loss of enzymatic activity. The concentration of LADH was checked spectrophotometrically employing an extinction coefficient, e, of 3.6x104 M-1cm- 1 at 280 nm. Enzymatic activity was measured by titration with NAD+ in excess pyrazole (Theorell and Yonetani, 1963). NAD+ and NADH solutions were obtained by dissolving each in buffer and assaying the concentration spectrophotometrically using extinction coefficients, e, of 1.78xl04 M-1cm- 1 at 260 nm and 6.22xIQ3 M-1cm- 1 at 340 nm, respectively. All reactions were carried out in 30 roM phosphate buffer solution. The ionic strength was maintained at 0.1 by adding Na2S04. Absorbance measurements were performed on a Varian Cary 210 spectrophotometer interfaced to an Apple lIe computer. Fast reactions were conducted on a Durrum-Gibson Model 1300 stopped-flow spectrophotometer interfaced to a NEC Powermate desktop computer with a DAS-20 (MetraByte) AID interface board. The 316
kinetic software was written in Turbo Pascal by the author. Fluorescence analysis of NADH produced by formaldehyde oxidation was done on a Perkin-Elmer 650-lOs fluorescence photometer. Unless specified otherwise, enzymatic reactions were initiated by injecting a small amount of enzyme solution into a pre-mixed coenzyme-substrate solution, and the change in NADH concentration monitored at 340 nm. Fast reactions with a half-life of a few seconds or less were performed on the stopped-flow equipment. For methanol and formaldehyde oxidation, the concentration change of formaldehyde during a reaction was also monitored with chromotropic acid by employing known procedures (Frisell, et al. 1954). Initial rates were obtained by fitting the data points to polynomial curves and extrapolating to zero time. Due to the fact that formaldehyde can be oxidized by NAD +, caution must be taken in devising product inhibition studies. For formaldehyde inhibition on methanol oxidation, low formaldehyde or NAD + concentrations were used so that the rate of oxidation of formaldehyde was small compared to the rate of methanol oxidation. In a few cases, the rate of formaldehyde oxidation had to be subtracted from the observed rate in order to obtain the initial rate of methanol oxidation. NAD + inhibition of formaldehyde reduction was studied using low NAD + concentrations so that the rate of formaldehyde oxidation was negligible compared to the rate of formaldehyde reduction.
RESULTS AND DISCUSSION 1.
METHANOL OXIDATION
The rate of methanol oxidation catalyzed by LADH is only about 3 % of the rate of ethanol oxidation. In the case of ethanol, extensive studies have shown that the reaction follows an ordered bi-bi mechanism (Wratten and Cleland, 1963; Pettersson, 1987). The characteristic of such a reaction mechanism is that there is a burst associated with the formation of the enzyme-NADH complex, which can be observed at 325 nm, followed by a slow dissociation of this complex. The burst of enzyme-NADH formation displays a deuterium isotope effect of about 6 and is much faster than the observed rate of free NADH formation (Brooks and Shore, 1971). Thus the dissociation of the enzyme-NADH complex is the rate-limiting step. In contrast, for the oxidation of methanol, no burst of enzyme-NADH was observed in our stopped-flow experiments. Instead, the observed result is the slow formation of free NADH. Using deuterated methanol, CD30H, the rate of free NADD formation is slower by a factor of about 5 compared to the rate of NADH formation from CH30H. These results are similar to those previously reported (Brook and Shore, 1971) and indicate that the hydride transfer step, that is, the conversion of the ternary complex, is rate-limiting for methanol oxidation. Product inhibition studies were performed, and results are shown in Figure 1 through Figure 4. When the NAD + concentration is varied, the product inhibition pattern is competitive for NADH as the product inhibitor and noncompetitive for formaldehyde. When the methanol concentration is varied, the product inhibition pattern is competitive for formaldehyde and noncompetitive for NADH. These patterns fit the rapid equilibrium random mechanism (Gulbinsky and Cleland, 1968). The kinetic parameters for the oxidation of methanol measured according to a rapid equilibrium random mechanism are listed in Table I. The kcat for methanol oxidation, which corresponds to the rate constant for conversion of the LADH-NAD + 317
1500
,.-..,
"......
~1000
~
0m ......,
C)
Q)
rn ......,
~ ~
~ ~
500
o
20
40
o
60
l/[Methanol] (lIM) Figure 1. Product inhibition of horse liver alcohol dehydrogenase by formaldehyde with methanol as varigble substrate. E~yme, 5.0xlO- M. NAD+, 5.00xlO M. Formaldehyde: (I) 0, (2) 2.03xlO-4M, (3) 4.88xl04 M.
l/[Methanol] (lIM) Figure 2. Product inhibition of horse liver alcohol dehydrogenase by NADH with methanol ~ variable substrat~ Enzyme, 2.3xlO- M. NAD+,5.70xlO M. NADH: (I) 0, (2) 2.94xlO~, (3) 5. 88xlO-6M.
1500~-.----------,
1500
'""'"
~
0Q) ~
~ ~
l/[NAD+] (lIM) Figure 3. Product inhibition of horse liver alcohol dehydrogenase by NADH with NAD + as variable substrate. Methanol, Enzyme, 2.3xlO-7 M. 3.10xlO-2M. NADH: 6 (I) 0, (2) 2.00xlO-6M, (3) 5.00xlO- M.
318
,.-.., ~ 1000
0Q) rn ......,
~ ~
500
o 5000 10000 l/[NAD+] (lIM) Figure 4. Product inhibition of horse liver alcohol dehydrogenase by formaldehyde with NAD+ as varigble substrate. E~me, 5.0xI0- M.
~)~,(~rl ~ij~~6~J:I(3r2~~~~ld~]~~:
methanol ternary complex, is about 30 times smaller than the reported value of 3.1 s-1 for ethanol oxidation (Theorell, et al., 1961). A kcat of 0.022 s-1 was measured (Table I) for the oxidation of CD30H, which is about 5 times smaller than that of CH30H, supporting the idea that the hydride transfer step is rate-limiting. The dissociation constant for the LADH-NAD+ complex obtained also agrees with the previously reported value (Sund and Theorell, 1963), in which a different technique was employed. The constants for the dissociation of methanol from the LADH-methanol (Kit» and the LADH-NAD+ -methanol complex (Kt» are very similar. This indicates that the binding of NAD + does not affect the binding of methanol greatly, and is different from the oxidation of ethanol and other primary alcohols where the binding of NAD+ promotes the binding of alcohol to the enzyme resulting in a preferred order mechanism. The similarity of Kib and % also indicates that deprotonation of methanol has not occurred during binding, because the positive charge of NAD + would have stabilized the zinc bound methoxide, thereby decreasing the value of Kb. For CD 30H oxidation, the dissociation constants for NAD+, Kia and Ka do not change significantly from those for CH 30H. Furthermore, the dissociation constants, Kib and K b , are similar for both CD30H and CH 30H. This is not surprising since the deuterated substrate should in principle only affect the hydride transfer step and not coenzyme or substrate binding to the enzyme. Pyrazole, an effective inhibitor of ethanol oxidation (Theorell and Yonetani, 1963), was used to inhibit the oxidation of methanol. The results show that pyrazole inhibits the reaction competitively with a Ki value of 3.5xlO-7 M. This is similar to the value of 3.9xlO-7 M obtained for pyrazole inhibition of ethanol oxidation (Pocker and Raymond, 1980). This inhibitor binds to the enzyme-NAD+ complex as an inner sphere ligand of the catalytic zinc (Shore and Gilleland, 1970) and thus blocks the substrate binding site. The close correspondence in inhibition patterns and inhibition constants noted for pyrazole in regard to the oxidation of both methanol and ethanol indicates that methanol binding to the catalytic zinc is similar to that of ethanol. The difference in the reaction mechanism and the magnitude of rate constants between methanol and ethanol can be explained by the hydrophobic interaction in the enzyme-NAD+ -alcohol complex. Within the alcohol binding site of LADH there are two binding regions, a hydroxyl binding region and a hydrophobic binding region (Dalziel and Dickinson, 1967). When a substrate binds to the enzyme, the interaction between the hydrophobic binding region and the hydrophobic chain on the substrate stabilizes substrate binding. Methanol, having a chain of only one carbon atom, cannot bind as ethanol and other primary alcohols to the hydrophobic region. Because the methyl group is not held in the proper orientation for hydride transfer, methanol exhibits both a weak binding constant and a slow turnover rate. The lack of a hydrophobic chain also accounts for the random mechanism for methanol oxidation. Early isotope exchange measurements, and analysis of substrate inhibition or activation patterns at high substrate concentration show that the LADH reaction can be generalized as a random mechanism in Scheme I (Branden, et al., 1975; Kamlayand Shore, 1983). However, for most alcohols and aldehydes, the ordered bi-bi mechanism (upper path in Scheme I) has been established over concentration ranges where Michaelis-Menten kinetics are obeyed. The preferred ordered mechanism for most substrates is followed because NAD+ binding to LADH results in a protein conformational change (Coates, et aI., 1977; Hardman, 1981). This conformational change alters the space within the hydrophobic and hydroxyl binding regions, stabilizing
319
substrate binding and greatly enhancing the upper pathway in Scheme I. In contrast, the alternation of the hydrophobic binding region, has little or no effect on methanol binding, and as a result, both the upper and the lower pathways in Scheme I are catalytically significant for methanol oxidation.
2.
REDUCTION OF FORMALDEHYDE
The rate of formaldehyde reduction is much higher than that of methanol oxidation but still much slower than the reduction of acetaldehyde. The results of product inhibition studies are shown in Figures 5 through 8. When the NADH concentration is varied, the product inhibition pattern is competitive for NAD+ and noncompetitive for methanol. When the formaldehyde concentration is varied, the product inhibition pattern is competitive for methanol and noncompetitive for NAD+. These patterns suggest that a rapid equilibrium random mechanism is followed. The constants calculated according to a rapid equilibrium random mechanism are listed in Table I. It is noted that the rate for the turnover of LADH-NADHformaldehyde complex is about 500 times larger than that of LADH-NAD + -methanol complex. The binding of formaldehyde has little or no effect on the observed binding constants of NADH. The value of the dissociation constant for the enzyme-NADH complex is in agreement with the value obtained by equilibrium studies
Y. Pocker and Hong Li Department of Chemistry University of Washington Seattle, WA 98195
INTRODUCTION Liver alcohol dehydrogenase (LADH) is a well-characterized protein. Both its primary and tertiary structures are known, as are some of the catalytic mechanisms, isozyme differences, evolutionary divergences and a number of enzymatic properties (J6rnvall, 1970; Branden, et aI., 1975; Klinman, 1981; Eklund and Branden, 1983; Pettersson, 1987; Pocker, 1989). In contrast, relatively little is known about its interaction with methanol, although there is extensive structural homology between this compound and ethanol. Compared with ethanol, methanol is an agent with a much more pronounced toxicity, notably, the ability to cause visual impairment (Jacobsen, et al., 1986). Moreover, there is increasing concern about the potential of formaldehyde, a metabolite of methanol, to act as a carcinogen (Clary, et al., 1983). In regard to its oxidation by LADH, methanol also shows a much slower reaction rate. There are significant differences in enzymatic behavior between methanol and other primary alcohols. Studies on the interaction of methanol with LADH will enable us to understand the biological behavior as well as the mechanism of methanol metabolism. Due to its relatively slow reaction rate as compared to ethanol, methanol was generally believed not to be a substrate of liver alcohol dehydrogenase (Winer, 1958; Wratten and Cleland, 1965). Later studies have shown that it is a substrate of alcohol dehydrogenases from both liver and yeast (Mani, et aI., 1970; Pocker, et aI., 1987a & 1987b). However, prior to the current study, no detailed rationale has been put forward for the enzymatic oxidation of methanol. The oxidation mechanism of ethanol and many other primary alcohols by LADH has been established to be an ordered bi-bi process (the upper pathway in Scheme I). That is to say, the binding of NAD+ precedes the binding of the alcohol with the ratelimiting step being the dissociation of NADH from the enzyme-NADH complex (Wratten and Cleland, 1963). For the oxidation of methanol, however, transient and
Enzymology and Molecular Biology of Carbonyl Metabolism 3 Edited by H. Weiner et al., Plenum Press, New York, 1990
315
steady-state kinetic studies with deuterated analogues have shown that the rate-limiting step is the hydride transfer in the enzyme-NAD+ -methanol complex (Brooks and Shore, 1971). A (EA)
Kia
P
B
Kp
Kb (EAB)
k
(EPQ)
r----E (EP)
(EB)
Ka B
Kiq
(EQ)
E
Kib
Q
A
Kip
Kq Q
P
SCHEME I. Random Mechanism for Liver Alcohol Dehydrogenase. E: LADH; A: NAD+; B: alcohol; P: aldehyde; Q: NADH.
The purpose of this study was to delineate, in detail, the kinetics and mechanism of the reactions of methanol and its metabolites with liver alcohol dehydrogenase. Our results indicate that methanol binds to a similar site in the enzyme as does ethanol, but due to the lack of an appreciable hydrophobic chain, it exhibits different kinetics and is the only simple primary alcohol found to follow a rapid equilibrium random mechanism (combined upper and lower pathways in Scheme I). EXPE~ENTALPROCEDURES
Horse LADH, NAD+, and NADH were obtained from Sigma Chemical Company. Pyrazole (recrystallized twice before use) was obtained from Aldrich Chemical Company. All other chemicals used were reagent grade. Methanol solutions were obtained by dissolving a weighed amount of absolute methanol in buffer. Formaldehyde solutions were prepared by distilling an aqueous solution of methenamine acidified with an equivalent amount of H2S04. The resulting distillate was neutralized, redistilled, and its concentration checked both gravimetrically with dimethylcyclohexanedione (Weinberger, 1931), and enzymatically using an excess of NADH in the presence of LADH. LADH, NAD+ and NADH solutions were prepared immediately before each experiment. The LADH solution was kept at OOC to prevent loss of enzymatic activity. The concentration of LADH was checked spectrophotometrically employing an extinction coefficient, e, of 3.6x104 M-1cm- 1 at 280 nm. Enzymatic activity was measured by titration with NAD+ in excess pyrazole (Theorell and Yonetani, 1963). NAD+ and NADH solutions were obtained by dissolving each in buffer and assaying the concentration spectrophotometrically using extinction coefficients, e, of 1.78xl04 M-1cm- 1 at 260 nm and 6.22xIQ3 M-1cm- 1 at 340 nm, respectively. All reactions were carried out in 30 roM phosphate buffer solution. The ionic strength was maintained at 0.1 by adding Na2S04. Absorbance measurements were performed on a Varian Cary 210 spectrophotometer interfaced to an Apple lIe computer. Fast reactions were conducted on a Durrum-Gibson Model 1300 stopped-flow spectrophotometer interfaced to a NEC Powermate desktop computer with a DAS-20 (MetraByte) AID interface board. The 316
kinetic software was written in Turbo Pascal by the author. Fluorescence analysis of NADH produced by formaldehyde oxidation was done on a Perkin-Elmer 650-lOs fluorescence photometer. Unless specified otherwise, enzymatic reactions were initiated by injecting a small amount of enzyme solution into a pre-mixed coenzyme-substrate solution, and the change in NADH concentration monitored at 340 nm. Fast reactions with a half-life of a few seconds or less were performed on the stopped-flow equipment. For methanol and formaldehyde oxidation, the concentration change of formaldehyde during a reaction was also monitored with chromotropic acid by employing known procedures (Frisell, et al. 1954). Initial rates were obtained by fitting the data points to polynomial curves and extrapolating to zero time. Due to the fact that formaldehyde can be oxidized by NAD +, caution must be taken in devising product inhibition studies. For formaldehyde inhibition on methanol oxidation, low formaldehyde or NAD + concentrations were used so that the rate of oxidation of formaldehyde was small compared to the rate of methanol oxidation. In a few cases, the rate of formaldehyde oxidation had to be subtracted from the observed rate in order to obtain the initial rate of methanol oxidation. NAD + inhibition of formaldehyde reduction was studied using low NAD + concentrations so that the rate of formaldehyde oxidation was negligible compared to the rate of formaldehyde reduction.
RESULTS AND DISCUSSION 1.
METHANOL OXIDATION
The rate of methanol oxidation catalyzed by LADH is only about 3 % of the rate of ethanol oxidation. In the case of ethanol, extensive studies have shown that the reaction follows an ordered bi-bi mechanism (Wratten and Cleland, 1963; Pettersson, 1987). The characteristic of such a reaction mechanism is that there is a burst associated with the formation of the enzyme-NADH complex, which can be observed at 325 nm, followed by a slow dissociation of this complex. The burst of enzyme-NADH formation displays a deuterium isotope effect of about 6 and is much faster than the observed rate of free NADH formation (Brooks and Shore, 1971). Thus the dissociation of the enzyme-NADH complex is the rate-limiting step. In contrast, for the oxidation of methanol, no burst of enzyme-NADH was observed in our stopped-flow experiments. Instead, the observed result is the slow formation of free NADH. Using deuterated methanol, CD30H, the rate of free NADD formation is slower by a factor of about 5 compared to the rate of NADH formation from CH30H. These results are similar to those previously reported (Brook and Shore, 1971) and indicate that the hydride transfer step, that is, the conversion of the ternary complex, is rate-limiting for methanol oxidation. Product inhibition studies were performed, and results are shown in Figure 1 through Figure 4. When the NAD + concentration is varied, the product inhibition pattern is competitive for NADH as the product inhibitor and noncompetitive for formaldehyde. When the methanol concentration is varied, the product inhibition pattern is competitive for formaldehyde and noncompetitive for NADH. These patterns fit the rapid equilibrium random mechanism (Gulbinsky and Cleland, 1968). The kinetic parameters for the oxidation of methanol measured according to a rapid equilibrium random mechanism are listed in Table I. The kcat for methanol oxidation, which corresponds to the rate constant for conversion of the LADH-NAD + 317
1500
,.-..,
"......
~1000
~
0m ......,
C)
Q)
rn ......,
~ ~
~ ~
500
o
20
40
o
60
l/[Methanol] (lIM) Figure 1. Product inhibition of horse liver alcohol dehydrogenase by formaldehyde with methanol as varigble substrate. E~yme, 5.0xlO- M. NAD+, 5.00xlO M. Formaldehyde: (I) 0, (2) 2.03xlO-4M, (3) 4.88xl04 M.
l/[Methanol] (lIM) Figure 2. Product inhibition of horse liver alcohol dehydrogenase by NADH with methanol ~ variable substrat~ Enzyme, 2.3xlO- M. NAD+,5.70xlO M. NADH: (I) 0, (2) 2.94xlO~, (3) 5. 88xlO-6M.
1500~-.----------,
1500
'""'"
~
0Q) ~
~ ~
l/[NAD+] (lIM) Figure 3. Product inhibition of horse liver alcohol dehydrogenase by NADH with NAD + as variable substrate. Methanol, Enzyme, 2.3xlO-7 M. 3.10xlO-2M. NADH: 6 (I) 0, (2) 2.00xlO-6M, (3) 5.00xlO- M.
318
,.-.., ~ 1000
0Q) rn ......,
~ ~
500
o 5000 10000 l/[NAD+] (lIM) Figure 4. Product inhibition of horse liver alcohol dehydrogenase by formaldehyde with NAD+ as varigble substrate. E~me, 5.0xI0- M.
~)~,(~rl ~ij~~6~J:I(3r2~~~~ld~]~~:
methanol ternary complex, is about 30 times smaller than the reported value of 3.1 s-1 for ethanol oxidation (Theorell, et al., 1961). A kcat of 0.022 s-1 was measured (Table I) for the oxidation of CD30H, which is about 5 times smaller than that of CH30H, supporting the idea that the hydride transfer step is rate-limiting. The dissociation constant for the LADH-NAD+ complex obtained also agrees with the previously reported value (Sund and Theorell, 1963), in which a different technique was employed. The constants for the dissociation of methanol from the LADH-methanol (Kit» and the LADH-NAD+ -methanol complex (Kt» are very similar. This indicates that the binding of NAD + does not affect the binding of methanol greatly, and is different from the oxidation of ethanol and other primary alcohols where the binding of NAD+ promotes the binding of alcohol to the enzyme resulting in a preferred order mechanism. The similarity of Kib and % also indicates that deprotonation of methanol has not occurred during binding, because the positive charge of NAD + would have stabilized the zinc bound methoxide, thereby decreasing the value of Kb. For CD 30H oxidation, the dissociation constants for NAD+, Kia and Ka do not change significantly from those for CH 30H. Furthermore, the dissociation constants, Kib and K b , are similar for both CD30H and CH 30H. This is not surprising since the deuterated substrate should in principle only affect the hydride transfer step and not coenzyme or substrate binding to the enzyme. Pyrazole, an effective inhibitor of ethanol oxidation (Theorell and Yonetani, 1963), was used to inhibit the oxidation of methanol. The results show that pyrazole inhibits the reaction competitively with a Ki value of 3.5xlO-7 M. This is similar to the value of 3.9xlO-7 M obtained for pyrazole inhibition of ethanol oxidation (Pocker and Raymond, 1980). This inhibitor binds to the enzyme-NAD+ complex as an inner sphere ligand of the catalytic zinc (Shore and Gilleland, 1970) and thus blocks the substrate binding site. The close correspondence in inhibition patterns and inhibition constants noted for pyrazole in regard to the oxidation of both methanol and ethanol indicates that methanol binding to the catalytic zinc is similar to that of ethanol. The difference in the reaction mechanism and the magnitude of rate constants between methanol and ethanol can be explained by the hydrophobic interaction in the enzyme-NAD+ -alcohol complex. Within the alcohol binding site of LADH there are two binding regions, a hydroxyl binding region and a hydrophobic binding region (Dalziel and Dickinson, 1967). When a substrate binds to the enzyme, the interaction between the hydrophobic binding region and the hydrophobic chain on the substrate stabilizes substrate binding. Methanol, having a chain of only one carbon atom, cannot bind as ethanol and other primary alcohols to the hydrophobic region. Because the methyl group is not held in the proper orientation for hydride transfer, methanol exhibits both a weak binding constant and a slow turnover rate. The lack of a hydrophobic chain also accounts for the random mechanism for methanol oxidation. Early isotope exchange measurements, and analysis of substrate inhibition or activation patterns at high substrate concentration show that the LADH reaction can be generalized as a random mechanism in Scheme I (Branden, et al., 1975; Kamlayand Shore, 1983). However, for most alcohols and aldehydes, the ordered bi-bi mechanism (upper path in Scheme I) has been established over concentration ranges where Michaelis-Menten kinetics are obeyed. The preferred ordered mechanism for most substrates is followed because NAD+ binding to LADH results in a protein conformational change (Coates, et aI., 1977; Hardman, 1981). This conformational change alters the space within the hydrophobic and hydroxyl binding regions, stabilizing
319
substrate binding and greatly enhancing the upper pathway in Scheme I. In contrast, the alternation of the hydrophobic binding region, has little or no effect on methanol binding, and as a result, both the upper and the lower pathways in Scheme I are catalytically significant for methanol oxidation.
2.
REDUCTION OF FORMALDEHYDE
The rate of formaldehyde reduction is much higher than that of methanol oxidation but still much slower than the reduction of acetaldehyde. The results of product inhibition studies are shown in Figures 5 through 8. When the NADH concentration is varied, the product inhibition pattern is competitive for NAD+ and noncompetitive for methanol. When the formaldehyde concentration is varied, the product inhibition pattern is competitive for methanol and noncompetitive for NAD+. These patterns suggest that a rapid equilibrium random mechanism is followed. The constants calculated according to a rapid equilibrium random mechanism are listed in Table I. It is noted that the rate for the turnover of LADH-NADHformaldehyde complex is about 500 times larger than that of LADH-NAD + -methanol complex. The binding of formaldehyde has little or no effect on the observed binding constants of NADH. The value of the dissociation constant for the enzyme-NADH complex is in agreement with the value obtained by equilibrium studies
