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Reactivity and stability of alcohol dehydrogenase below 0° in alcohol-water solvents
406
SHORT COMMUNICATIONS
each reactant to the active site by means of an anionic charge: ionized carboxyl in the case of tyrosine; ionized phosphate group in the case of the coenzyme; and ionized carboxyl in the case of a-ketoglutarate. However, direct evidence is required to substantiate this concept. A study of pH and stability of the partially purified enzyme produced some surprising results as shown in Fig. I. In contrast to the sharp fall in activity in the pH-activity dependence curve on the acid side of pH 7.6, a pH-stability curve showed that resulting activity was nearly as great at pH values as low as pH 5.6 and below as it was at the pH optimum for activity under these conditions. This observation has been used recently to great advantage in increasing the recovery of enzymatic activity from columns of polyacrylamide gel3. This research was supported by research grants AM-o835o and CA-o7174, from the National Institute of Arthritis and Metabolic Diseases and the National Cancer Institute, National Institutes of Health, U.S. Public Health Service. G.L. is a Research Career Development Awardee, 3-K3-AM-I6,568 from the National Institute of Arthritis and Metabolic Diseases.
Fels Research Institute, Temple University School of Medicine, Philadelphia, Pa. (U.S.A.) I
2 3 4 5 6 7 8
Z. E. G. G. G. H. G. T.
GERALD L I T W A C K
ILGA WlNICOV M. SQUIRES
JOAN
N. CANELLAKIS AND P. P. COHEN, J. Biol. Chem., 222 (1956) 63. C. C. LIN, B. M. PITT, M. CIVEN AND W. E. KNOX, J. Biol. Chem., 233 (1958) 668. LITWACK AND I. WINICOV, s u b m i t t e d for publication. A. JACOBY AND B. N. LA DO, J. Biol. Chem., 239 (1964) 419. LITWACK, Biochim. Biophys. Acta, 56 (1962) 593. LINEWEAVER AND D. BURK, J. Am. Chem. Sue., 56 (1934) 658. LITWACK, J. Biol. Chem., 228 (1957) 823. I. DIAMONDSTONE AND G'. LITWACK, j . Biol. Chem., 238 (1963) 3859 •
Received July i4th, 1966 Biochim. Biophys. Acta, 128 (1966) 404-406
BBA 63209
Reactivity and stability of alcohol dehydrogenase below O* in alcohol-water solvents The present experiments with yeast alcohol dehydrogenase (EC i.I.I.I) are a continuation of the studies on enzyme reactions at low temperatures initiated with the proteolytic enzyme, a-chymotrypsinl, *. In general the methods previously used and described in detail were also found applicable to the hydrogenase. The reactions of alcohol dehydrogenase were followed by the fluorescence of NADH at 45 ° m/t, when excited at 34 ° m/~, and of alcohol dehydrogenase at 34 ° m# excited at 290 in/,. * T h e p u r p o s e of such experiments h a s b e e n described b y S. FREED ~ in a n article " C h e m i c a l - b i o c h e m i c a l signal a n d noise".
Biochim. Biophys. Acta, 128 (1966) 406-409
SHORT COMMUNICATIONS
407
The solutions were prepared first b y dissolving alcohol dehydrogenase (Worthington Biochemical Corp.) in aqueous Tris buffer (pH 7, I 0.05) at o ° and then injecting this solution with a micropipette as a spray into a buffered ethanol-methanol-water mixture precooled to the low temperature. When the alcoholic mixture was at --80 °, the injected aqueous solution of the enzyme formed small ice crystals which dissolved slowly, but more rapidly with vibration. The effect of alcohol upon the activity of alcohol dehydrogenase was determined at o °. The solution of NAD + was added by injection and the increase in the fluorescence intensity at 450 m/z was followed with time. The initial concentrations of NAD + and alcohol dehydrogenase were, respectively, 2.5" lO-4 M and 5" IO-S M. Table I gives the initial rates of formation of NADH as a function of the concentration of alcohol (methanol-ethanol, 2 : I, by vol.). In alcoholic solutions, the magnitude of the pH denoted here is the pH produced by the same quantities of Tris and HC1 in an equal volume of water. Inhibition b y alcohol up to at least 77% proved to be reversible at o ° upon addition of water. Incubation experiments were performed by injecting aqueous solutions of alcohol dehydrogenase at o ° into absolute alcohols at --80 ° so that the final compositions of the two solvents water-methanol-ethanol were 5:45:50 and 4.8:31.7: 63.5, b y vol. Incubation proceeded for 20 days at --80 °. Aliquots were withdrawn daily and tested for enzymic activity by injecting 125/~1 in a cold pipette into 4 ml buffered aqueous solutions of NAD+ (pH 9, I 0.05) at o °. No denaturation was detected in either solvent. No trend of activity with time was observed within the experimental variation of lO%. In the light of the stability of alcohol dehydrogenase toward alcohols at o °, initial rates of enzymic oxidation were determined as a function of temperature from o ° to --3 o°. The solvent was composed of water-methanol-ethanol (45:35:20, b y vol.) NAD + I.O.lO -4 and alcohol dehydrogenase 1.2-IO -~ M (pH 9.0, I 0.05). The rates were determined in absolute units by calibration of fluorescence intensity at 450 m# with known concentration of NADH (Boeringer, Mannheim). Fig. I illustrates the results. Because of the sensitivity of the fluorometric method, the increase in concentration of NADH at -3 °0 could be observed in minutes. These rates indicate a single activation energy of 27 -4- 2.5 kcal/mole as given in the figure. The precision of this value indicated that little change in the conformation of the protein molecule occurred over this temperature range. In contrast, a-chymotrypsin exhibited a rather abrupt increase in activation energy below about --18 ° While the enzymic oxidation would be exceedingly slow at --80 ° , an intermediate reaction, the combination of alcohol dehydrogenase with NADH proved to be virtually instantaneous at this temperature. The formation of the complex, enzyme-coenzyme, was followed by changes in fluorescence accompanying energy transfer similar to numerous other observations 3-s on hydrogenases in aqueous solution where the intensity of fluorescence of the dehydrogenase had been quenched at 340 m/* and that of NADH augmented with a concurrent shift of the maximum to 448 m/z. In the present study the injection of ioo/zl of a solution of N A D H near --80 ° into 4 ml of the solution of alcohol dehydrogenase at -- 80 ° immediately quenched the fluorescence of the latter at 34 ° m/, and increased the fluorescence intensity at Biochim. Biophys. Aaa, 128 (1966) 406-409
408
SHORT COMMUNICATIONS
I I t I I N~=2ZO::t: 2.5kCQIM -1
10-8"~-~ ~ 0 10-S---
N~O
~:710-11 ..-., 3.6
I 3.7
I x8
I 3.9
I ~o
I 4.1
45
1/r l o 3
Fig. i. Activity of alcohol dehydrogenase as function of the t e m p e r a t u r e m e a s u r e d b y the rate of production of N A D H . Alcohol dehydrogenase, 1 . 2 . i o - S M ; NAD+, i . i o - * M ; Tris buffer (pH 7, I 0.05). Solvent, w a t e r - m e t h a n o l - e t h a n o l (45:35:2o, b y vol.).
450 m#. The peak of the fluorescence of N A D H in the alcoholic solutions in the absence of the enzyme was at 450 rn~ and this m a x i m u m underwent little shift in its wavelength on complex formation. The increase in the intensity at 450 m# was 20% when the the final concentration of N A D H was 5 "1o-6 M and that of alcohol dehydrogenase, 1.25" lO -6 M (pH 9, I 0.50 ) and when the composition of the final solvent was water-methanol-ethanol (2.5:48.75:48.75, b y vol.). Similarly, in exploratory experiments there was immediate quenching of the fluorescence of alcohol dehydrogenase b y N A D H representing complex formation at --123 °. An alcoholic solution of N A D H at --80 °, IOO #1 in volume, was injected into 4 ml of enzyme solution at -- 123 ° with the final concentration of the former 5" lO-7 M and that of the latter 1.25" lO -7 M. The solvent had been made more fluid b y condensing the TABLE I E N Z Y M A T I C A C T I V I T Y OF A L C O H O L D E H Y D R O G E N A S F ~ AS A F U N C T I O N AT 0 °
OF ALCOHOL CONCENTRATION
"Iris buffer (pH 9, I o.05) ; alcohol dehydrogenase, 5 "Io-S M; NAD+, 2.5" lO -4 M Volume percent Methanol
Ethanol
Total alcohol
Water
4 12"3 20,7 26,7 29,o 3o.o
8 24.7 41.4 53.4 58.o 6o.0
I2 37 62.I 8o.1 87 9o
88 63 37,9 I9,9 13,o IO
Rate of formation NA DH ( M . sec -x ) 9.65" 9.5 ° . 3-49' 1.6o. 5.oo, o
Biockim. Biophys. Acta, 128 (1966) 406-4o9
IO-? I o-? lO-7 i o -7 io-9
409
SHORT COMMUNICATIONS
normally gaseous substances, dimethyl ether and ethylene oxide, into a mixture of the alcohols and water at about --80 ° and the resulting liquid was then cooled to - - 1 2 3 °. The final composition by volume was water-methanol-ethano]-dimethyl ether-ethylene oxide (I.I :5:5:5:5, by vol.). A further reduction in temperature seems to be required before the formation of the enzyme-coenzyme complex becomes sufficiently slow for measurement of the rate b y simple means. Retardation by alcohol may not be as effective in the formation of the complex as in the overall enzymatic reaction. The rate of the enzymic reaction at o ° was about two hundred times slower in 87% than in 14% alcohol (see Table I). This rearch was performed under the auspices of the U.S. Atomic Energy Commission.
Chemistry Department, Brookhaven National Laboratory, Upton, New York, N.Y., (U.S.A.) I 2 3 4 5
BENON H. J. BIELSKI ROBERT HENRETIG* SIMON F R E E D * *
B. H. J. BIELSKI AND S. FREED, Biochim. Biophys. Acta, 89 (1964) 314. S. FREED, Science ,5 o (1965) 576. P. n . BOYER ANn a . THEORELL, Acta Chem. Scand., i o (1956) 447. S. F. VELICK, J. Biol. Chem., 233 (I958) 1455. A. D. WINER, C*. W. SCHWERT AND D. B. S. MILLAR, J. Biol. Chem., 234 (1959) 1149.
Received March 4th, 1966 Revised manuscript received July 22nd, 1966 * P r e s e n t a d d r e s s : S t a n f o r d U n i v e r s i t y , Palo Alto, Calif., U.S.A. ** P r e s e n t a d d r e s s : M o u n t Sinai Medical a n d G r a d u a t e Schools, N e w York, N.Y., U.S.A.
Biochim. Biophys. Acta, 128 (1966) 4 0 7 - 4 0 9
SHORT COMMUNICATIONS
each reactant to the active site by means of an anionic charge: ionized carboxyl in the case of tyrosine; ionized phosphate group in the case of the coenzyme; and ionized carboxyl in the case of a-ketoglutarate. However, direct evidence is required to substantiate this concept. A study of pH and stability of the partially purified enzyme produced some surprising results as shown in Fig. I. In contrast to the sharp fall in activity in the pH-activity dependence curve on the acid side of pH 7.6, a pH-stability curve showed that resulting activity was nearly as great at pH values as low as pH 5.6 and below as it was at the pH optimum for activity under these conditions. This observation has been used recently to great advantage in increasing the recovery of enzymatic activity from columns of polyacrylamide gel3. This research was supported by research grants AM-o835o and CA-o7174, from the National Institute of Arthritis and Metabolic Diseases and the National Cancer Institute, National Institutes of Health, U.S. Public Health Service. G.L. is a Research Career Development Awardee, 3-K3-AM-I6,568 from the National Institute of Arthritis and Metabolic Diseases.
Fels Research Institute, Temple University School of Medicine, Philadelphia, Pa. (U.S.A.) I
2 3 4 5 6 7 8
Z. E. G. G. G. H. G. T.
GERALD L I T W A C K
ILGA WlNICOV M. SQUIRES
JOAN
N. CANELLAKIS AND P. P. COHEN, J. Biol. Chem., 222 (1956) 63. C. C. LIN, B. M. PITT, M. CIVEN AND W. E. KNOX, J. Biol. Chem., 233 (1958) 668. LITWACK AND I. WINICOV, s u b m i t t e d for publication. A. JACOBY AND B. N. LA DO, J. Biol. Chem., 239 (1964) 419. LITWACK, Biochim. Biophys. Acta, 56 (1962) 593. LINEWEAVER AND D. BURK, J. Am. Chem. Sue., 56 (1934) 658. LITWACK, J. Biol. Chem., 228 (1957) 823. I. DIAMONDSTONE AND G'. LITWACK, j . Biol. Chem., 238 (1963) 3859 •
Received July i4th, 1966 Biochim. Biophys. Acta, 128 (1966) 404-406
BBA 63209
Reactivity and stability of alcohol dehydrogenase below O* in alcohol-water solvents The present experiments with yeast alcohol dehydrogenase (EC i.I.I.I) are a continuation of the studies on enzyme reactions at low temperatures initiated with the proteolytic enzyme, a-chymotrypsinl, *. In general the methods previously used and described in detail were also found applicable to the hydrogenase. The reactions of alcohol dehydrogenase were followed by the fluorescence of NADH at 45 ° m/t, when excited at 34 ° m/~, and of alcohol dehydrogenase at 34 ° m# excited at 290 in/,. * T h e p u r p o s e of such experiments h a s b e e n described b y S. FREED ~ in a n article " C h e m i c a l - b i o c h e m i c a l signal a n d noise".
Biochim. Biophys. Acta, 128 (1966) 406-409
SHORT COMMUNICATIONS
407
The solutions were prepared first b y dissolving alcohol dehydrogenase (Worthington Biochemical Corp.) in aqueous Tris buffer (pH 7, I 0.05) at o ° and then injecting this solution with a micropipette as a spray into a buffered ethanol-methanol-water mixture precooled to the low temperature. When the alcoholic mixture was at --80 °, the injected aqueous solution of the enzyme formed small ice crystals which dissolved slowly, but more rapidly with vibration. The effect of alcohol upon the activity of alcohol dehydrogenase was determined at o °. The solution of NAD + was added by injection and the increase in the fluorescence intensity at 450 m/z was followed with time. The initial concentrations of NAD + and alcohol dehydrogenase were, respectively, 2.5" lO-4 M and 5" IO-S M. Table I gives the initial rates of formation of NADH as a function of the concentration of alcohol (methanol-ethanol, 2 : I, by vol.). In alcoholic solutions, the magnitude of the pH denoted here is the pH produced by the same quantities of Tris and HC1 in an equal volume of water. Inhibition b y alcohol up to at least 77% proved to be reversible at o ° upon addition of water. Incubation experiments were performed by injecting aqueous solutions of alcohol dehydrogenase at o ° into absolute alcohols at --80 ° so that the final compositions of the two solvents water-methanol-ethanol were 5:45:50 and 4.8:31.7: 63.5, b y vol. Incubation proceeded for 20 days at --80 °. Aliquots were withdrawn daily and tested for enzymic activity by injecting 125/~1 in a cold pipette into 4 ml buffered aqueous solutions of NAD+ (pH 9, I 0.05) at o °. No denaturation was detected in either solvent. No trend of activity with time was observed within the experimental variation of lO%. In the light of the stability of alcohol dehydrogenase toward alcohols at o °, initial rates of enzymic oxidation were determined as a function of temperature from o ° to --3 o°. The solvent was composed of water-methanol-ethanol (45:35:20, b y vol.) NAD + I.O.lO -4 and alcohol dehydrogenase 1.2-IO -~ M (pH 9.0, I 0.05). The rates were determined in absolute units by calibration of fluorescence intensity at 450 m# with known concentration of NADH (Boeringer, Mannheim). Fig. I illustrates the results. Because of the sensitivity of the fluorometric method, the increase in concentration of NADH at -3 °0 could be observed in minutes. These rates indicate a single activation energy of 27 -4- 2.5 kcal/mole as given in the figure. The precision of this value indicated that little change in the conformation of the protein molecule occurred over this temperature range. In contrast, a-chymotrypsin exhibited a rather abrupt increase in activation energy below about --18 ° While the enzymic oxidation would be exceedingly slow at --80 ° , an intermediate reaction, the combination of alcohol dehydrogenase with NADH proved to be virtually instantaneous at this temperature. The formation of the complex, enzyme-coenzyme, was followed by changes in fluorescence accompanying energy transfer similar to numerous other observations 3-s on hydrogenases in aqueous solution where the intensity of fluorescence of the dehydrogenase had been quenched at 340 m/* and that of NADH augmented with a concurrent shift of the maximum to 448 m/z. In the present study the injection of ioo/zl of a solution of N A D H near --80 ° into 4 ml of the solution of alcohol dehydrogenase at -- 80 ° immediately quenched the fluorescence of the latter at 34 ° m/, and increased the fluorescence intensity at Biochim. Biophys. Aaa, 128 (1966) 406-409
408
SHORT COMMUNICATIONS
I I t I I N~=2ZO::t: 2.5kCQIM -1
10-8"~-~ ~ 0 10-S---
N~O
~:710-11 ..-., 3.6
I 3.7
I x8
I 3.9
I ~o
I 4.1
45
1/r l o 3
Fig. i. Activity of alcohol dehydrogenase as function of the t e m p e r a t u r e m e a s u r e d b y the rate of production of N A D H . Alcohol dehydrogenase, 1 . 2 . i o - S M ; NAD+, i . i o - * M ; Tris buffer (pH 7, I 0.05). Solvent, w a t e r - m e t h a n o l - e t h a n o l (45:35:2o, b y vol.).
450 m#. The peak of the fluorescence of N A D H in the alcoholic solutions in the absence of the enzyme was at 450 rn~ and this m a x i m u m underwent little shift in its wavelength on complex formation. The increase in the intensity at 450 m# was 20% when the the final concentration of N A D H was 5 "1o-6 M and that of alcohol dehydrogenase, 1.25" lO -6 M (pH 9, I 0.50 ) and when the composition of the final solvent was water-methanol-ethanol (2.5:48.75:48.75, b y vol.). Similarly, in exploratory experiments there was immediate quenching of the fluorescence of alcohol dehydrogenase b y N A D H representing complex formation at --123 °. An alcoholic solution of N A D H at --80 °, IOO #1 in volume, was injected into 4 ml of enzyme solution at -- 123 ° with the final concentration of the former 5" lO-7 M and that of the latter 1.25" lO -7 M. The solvent had been made more fluid b y condensing the TABLE I E N Z Y M A T I C A C T I V I T Y OF A L C O H O L D E H Y D R O G E N A S F ~ AS A F U N C T I O N AT 0 °
OF ALCOHOL CONCENTRATION
"Iris buffer (pH 9, I o.05) ; alcohol dehydrogenase, 5 "Io-S M; NAD+, 2.5" lO -4 M Volume percent Methanol
Ethanol
Total alcohol
Water
4 12"3 20,7 26,7 29,o 3o.o
8 24.7 41.4 53.4 58.o 6o.0
I2 37 62.I 8o.1 87 9o
88 63 37,9 I9,9 13,o IO
Rate of formation NA DH ( M . sec -x ) 9.65" 9.5 ° . 3-49' 1.6o. 5.oo, o
Biockim. Biophys. Acta, 128 (1966) 406-4o9
IO-? I o-? lO-7 i o -7 io-9
409
SHORT COMMUNICATIONS
normally gaseous substances, dimethyl ether and ethylene oxide, into a mixture of the alcohols and water at about --80 ° and the resulting liquid was then cooled to - - 1 2 3 °. The final composition by volume was water-methanol-ethano]-dimethyl ether-ethylene oxide (I.I :5:5:5:5, by vol.). A further reduction in temperature seems to be required before the formation of the enzyme-coenzyme complex becomes sufficiently slow for measurement of the rate b y simple means. Retardation by alcohol may not be as effective in the formation of the complex as in the overall enzymatic reaction. The rate of the enzymic reaction at o ° was about two hundred times slower in 87% than in 14% alcohol (see Table I). This rearch was performed under the auspices of the U.S. Atomic Energy Commission.
Chemistry Department, Brookhaven National Laboratory, Upton, New York, N.Y., (U.S.A.) I 2 3 4 5
BENON H. J. BIELSKI ROBERT HENRETIG* SIMON F R E E D * *
B. H. J. BIELSKI AND S. FREED, Biochim. Biophys. Acta, 89 (1964) 314. S. FREED, Science ,5 o (1965) 576. P. n . BOYER ANn a . THEORELL, Acta Chem. Scand., i o (1956) 447. S. F. VELICK, J. Biol. Chem., 233 (I958) 1455. A. D. WINER, C*. W. SCHWERT AND D. B. S. MILLAR, J. Biol. Chem., 234 (1959) 1149.
Received March 4th, 1966 Revised manuscript received July 22nd, 1966 * P r e s e n t a d d r e s s : S t a n f o r d U n i v e r s i t y , Palo Alto, Calif., U.S.A. ** P r e s e n t a d d r e s s : M o u n t Sinai Medical a n d G r a d u a t e Schools, N e w York, N.Y., U.S.A.
Biochim. Biophys. Acta, 128 (1966) 4 0 7 - 4 0 9