Kinetics and Dissociation Constants of Liver Alcohol Dehydrogenase with 3-Acetyl Pyridine NAD+ and NADH

Transcription

1 European J. Biochem. 2 (19G7) Kinetics and Dissociation Constants of Liver Alcohol Dehydrogenase with 3-Acetyl Pyridine NAD+ and NADH J. D. SHORE and H. THEORELL Biochemistry Department, Nobel Medical Institute, Stockholm (Received February 9, 1967) Binary complex dissociation constants and kinetic di values of liver alcohol dehydrogenase were determined for 3-acetyl pyridine NAD' and NADH. The reduced analog was bound 24 times less tightly than NADH to liver alcohol dehydrogenase whereas no significant difference was found between the binding of 3-acetyl pyridine NAD' and NAD' to liver alcohol dehydrogenate. The rates of interaction of substrates with binary complexes were one tenth as fast when the acetyl pyridine analogs were used. These results were interpreted with reference to the significance of thc pyridine ring amide group in binding, and the interaction of binary complexes with substrates. The reaction mechanism of horse liver alcohol dehydrogenase has been studied extensively by Theorell and Chance [l 1, Dalziel [2], and Theorell and McKinley-McKee [3]. It is now generally agreed that, from a kinetic point of view, the sequence is ordered with the first step being binding of coenzyme and the last step dissociation of coenzyme. Several recent publications [4,5] indicate that the enzyme can bind substrate first but this reaction is not kinetically significant. The binding of pyridine nucleotide coenzymes to the LADH has also been thoroughly investigated [6,7], indicating that NADH is much more tightly bound than NAD+. It had previously been postulated [S] that the amino group and the nitrogen atom in position 9 of the adenine ring of NAD' and NADH formed a bidentate chelate with the Zn atom at the active center of LADH. Subsequent studies by Yonetani [9] and Yonetani and Theorell [lo] demonstrated that it was possible to create an o-phenanthroline-enzyme-adpr complex, and that the NAD' competitive inhibitions by ADPR and o-phenanthroline are additive. It therefore now seems probable that the ADPR part of the NAD+ is not involved in the formation of a bidentate Zn chelate. The fact that o-phenanthroline is an inhibitor competitive with NAD+ and NADH [ill indicates that the pyridine ring must interact with Zn. The 3-acetyl-pyridine analog of NAD' was first prepared by Kaplan and Ciotti [i2]. Subsequent studies indicated that the activity of this analog Abbreviations used. E or LADH, horse liver alcohol dehydrogenase (EC ); or NADf, oxidized nicotinaniide adenine dinucleotide; R or NADH, reduced nicotinamide adenine dinucleotide; 3-AP, 3-acetyl pyridinc. with LADH was much faster than with NAD' under standard assay conditions at high alcohol concentrations [13], and that the binding of the reduced analog to LADH is accompanied by spectral [I41 and fluorescence emission [I51 shifts. The purpose of this study is to investigate the kinetics and equilibria of the LADH reaction using 3-acetyl-pyridine analogs of NAD' and NADH as coenzymes. Since the sequences of the LADH reactions with ordinary NAD' and NADH are fairly well established, and the steady-state equations describing them are known, the significance of the amide group on the pyridine ring in binding of the coenzyme can be to some extent evaluated. METHODS LADH (F,) was prepared according to Dalziel[16] and assayed by the method of Dalziel [17], based on an A,,, of.455 ml/mg xcm-l for pure enzyme. NAD' (), NADH (R) and 3-acetyl-pyridine NAD' were obtained from Sigma Chemical Company. The NAD' was repurified by the method of Dalziel [la]. 3-Acetyl-pyridine NADH was made by the method of Rafter and Colowick [19]. The fluorometric method of Theorell and McKinley-McKee [3] was used for kinetic experiments, with the exception that a commercial stabilized power supply (Oltronix), was used with the photomultiplier of the fluorometer. The binding of 3-acetyl-pyridine NADH and 3-acetylpyridine NAD' was studied by the method of Theorell and iner [6] using a spectrophotofluorometer. All kinetic and binding studies were carried out at ph 7. in phosphate buffer (.1 ionic strength) at 23".

2 Vol.2, No.1, 1967 J. D. SHORE and H. THEORELL 33 RESULTS Binding of the Analog to LADH. Fig.1 demonstrated the shift in emission spectrum obtained when 3-acetyl-pyridine NADH is bound to LADH. The excitation wavelength was 35 mp and the emission spectrum shifted from a peak at 495 mp to one at 47 mp. hen corrected for the wavelength dependence of our photomultiplier sensitivity, these values become around 48 and 455 mp. These are somewhat higher than the values 47 and 44mp given by equilibrium measurements and kinetically from the Theorell-Chance steady-state equation. This correlation is indicative of the ordered nature of the reaction sequence. >- t cn z w b- z z m LT 3 L L AVELENGTH (mp) Fig.l.'Emission spectra of 3-AP NADH and 3-AP NADH- LADH complex. Excitation at 35mp in ph 7.,.1 p phosphat buffer. (a) 2.5 pn 3-AP NADH; (b) 2.5 yn 3-AP NADH plus 11.5 pn LADH Shifrin et al. [13]. These authors, however, do not state whether their values were corrected or not. By measuring changes in fluorescence at 45 mp obtained by adding 3-acetyl-pyridine NADH to LADH, we were able to calculate that KE,R was = 4.8 pm and KE,O ranged from 6 to 12 pm. Determination Values. values were determined by the method of Dalziel [2], varying ethanol at 5 fixed concentrations of 3-acetylpyridine NAD* and varying acetaldehyde at 5 fixed concentrations of 3-acetyl-pyridine NADH. The primary and secondary plots are presented in Fig.2 through 7. The numerical values obtained for values can be found in Table 2. Pit to Theorell-Chance Mechanism. In order to investigate whether the LADH reaction can still be kinetically described by the Theorell-Chance mechanism with 3-acetyl-pyridine coenzyme, values, and dissociation and equilibrium constants, were fitted to the relationships proposed by Dalziel[2]. This fit can be seen in Table 1. The most significant aspect of this is the correlation between KE,R and KE,O values determined directly from 3 European J. Biochem., Vol VCACETALDEHYDEI (mm-') Fig.2. Primary plots: Vuriation of reciprocal of initial rate with reciprocal of aldehyde concentration for several 3-AP NADH concentrations. e represents pn enzyme concentration, and the units of elv are in seconds. A, 8.1 pm; n, 4.5 pm;, 2.4 pm;, 1.6 pm.4 O'_I O! I * /13-AP NADHl (pm-') Fig. 3. Secondary plot of intercepts frma Fig. 2 us. reciprocal 3-AP NADH concentration. The units of the ordinate are seconds Comparison between NAB-NABH and the 3- Acetyl-pyridine Analogs. In Table 2 values and dissociation constants for the NAD-NADH coenzymes and the 3-acetyl-pyridine analogs are listed. The

3 34 Liver Alcohol Dehydrogenase Reaction with 3-Acetyl Pyridine NADf European J. Biochem. importance of the amide group of the pyridine ring in both binding and transfer of electrons can be seen from this comparison. 1 correlations with the equilibrium constant, are not unexpected since considerable error may be present in the QI2 and values, and the ratio of multiplied cn 6 Id a 9 cn 4 f.15 w IL I- z.ic 2 n! l/c3-ap NADHl (pm-') Pig.4. Secondary plot of slopes from Fig.2 us. reciprocal 3-AP NADH concentration. The units of the ordinate are mm seconds O L, I ( 1/[3-AP NAD'I (pm-') Fig. 6. Secondary plot of intercepts from Fig. 5 vs. reciprocal of 3-AP NAD+ eoncentration. The units of the ordinate are seconds / 4 I I.2.4 Q METHANOL1 (rnmt1) Fig.5. Primary plots: Variation of reciprocal of initial rate with reciprocal of ethanol concentration for several 3-AP NAD+ concentrations. e represents pn enzyme concentration, and the units of C/V are in seconds. A, 17 pm; e, 68 pm;, 34 pm; A, 27 pm; 24 pm DISCUSSION The values listed in Table 1 indicate that the mechanism is the same with 3-AP NAD' and 3-AP NADH as it is when NAD' and NADH are the coenzymes. The deviations found, particularly for ( 5 1/[3-AP NAD'I (pm-') Fig.7. Secondary plot of slopes from Fig.5 us. reciprocal of 3-AP NAD+ concentration. The units of the ordinate are values. The most significant correlation, between the dissociation constants of the binary complexes determined directly and kinetically, is within the realm of expectations. The range in values for KE, determined directly is probably due to enzyme-bound alcohol, which it was not possible to

4 ~ ~~ ~~ V1.2, No. 1, D. SHORE and H. THEORELL remove at the time. It seems probable, therefore, that the enzyme mechanism with the 3-acetylpyridine analog can still be kinetically described in the manner indicated by Theorell and Chance [l], as an ordered reaction in which interconversion and dissociation of ternary complexes is not rate limiting. Table 1. Fit of kinetic values and directly determined equilibrium constants for 3-acetyl-pyridine NAD+ and NADH to the Theorell-Chance mechanism at ph 7. Einetic relationship Corrclated value % = 1.4~1-~ K~~~ii. = =.4 sec =.5 sec 6.25 pm KX,R = 4.8 pm =5pM Kg,o = 6-12 pm Table 2. Comparison of kinetic and equilibrium values obtained with NAD+-NADH and the 3-acetyl-pyridine analogs Measurements are made in phosphate buffer, ph 7. (.1 ionic strength) Kinetic or equilibrium NAD+ - NADH 3-AP NADf - 3-AP NADH cons tan t KE~w. 9 x 1-5 KE,B.2 pm Kn,o 133 see x 5.1 sec x sec 2. sec x pm 94 sec x %!I% 3 x [13] sec.25 sec x pm 5. sec x pm.4 sec 2.5 sec x NM 85 sec x'pm 17. The most striking aspect of the comparison listed in Table 2 is the difference between KE,R for NADH and for the 3-acetyl-pyridine analog. The amide group on the pyridine ring of NADH is apparently essential to binding, and its modification results in a dissociation constant 24 times higher. This explains the observation [13] that the 3-acetyl-pyridine analog is much more active, since the rate of dissociation of R from ER will determine activity when alcohol is the substrate at conditions near Vmax. Actually the rate of dissociation of ER, l/@o' is 7 times higher for 3-AP NADH than for NADH. The failure to find substrate inhibition at high ethanol concentrations [13] with 3-AP NAD' is also explained by the high KE,R value. The tendency for ER to break down is high enough to prevent the formation of abortive ER alcohol complex unless extremely high ethanol concentrations are used. The fact that no difference was found with the KE,O value for NAD+ and the 3-acetyl-pyridine analog was also of interest. The amide group of the pyridine ring of oxidized coenzyme is apparently not bound to the enzyme, perhaps due to repulsion of the positively charged ring. This might also explain the much tighter binding of reduced coenzyme to enzyme, as contrasted with oxidized coenzyme. The differences found in the Q2 values with 3-acetyl-pyridine coenzyme are also marked but rather difficult to interpret. Much evidence has accrued recently indicating that ternary coniplexes are formed, even though they are not kinetically significant. It is thus possible to express the substrate utilizing steps of the LADH mechanism in the following way : k k-2 k ' 1c-, ER + Ald 2_\ ER Ald F+ EOAlc EO -1 Alc. According to Dalziel [ZO], a mechanism such as this one would result in: and therefore : k,k-, 4 k,k + 2, b-,k'k-, Considering the complexity of the expressions * it is difficult to clearly delineate the rate constant or constants which are changed when the coenzyme analog is used. The unchanged ratio, = 18.4 for NAD+-NADH and 8515 = 17 for APNAD+-APNADH however, indicates that the rate limiting steps in both the forward and reverse reactions are changed to the same extent. Further work, using product inhibition, might provide a better indication of the actual rate constants which are lowered. The present study provides some knowledge of the importance of the pyridine ring amide group in the binding of NAD' and NADH to LADH, with inferences regarding the attraction of the oxidized and reduced pyridine ring to the enzyme and the interaction of binary enzyme-coenzyme complexes with substrates. Additional investigations currently in progress, using inhibitors and modified enzyme, are aimed towards elucidating the groups on the enzyme which interact with the amide group of the pyridine ring. 3'

5 36 SHORE and THEORELL: Liver Alcohol Dehydrogenase Reaction with 3-Acetyl Pyridine NADf European J. Biochem. This work was supported by a post-doctoral fellowship to J. D. S. from the Muscular Dystrophy Associations of America, Inc., and research grants from the Swedish State Medical Research Council and Institutet for Maltdrycksforskning. The technical assistance of Mrs. C. Popper is gratefully acknowledged. REFERENCES 1. Theorell, H., and Chance, B., Actu Chem. Scand. 5 (1951) Dalziel, K., J. Biol. Chem. 238 (1963) Theorell. H.. and McKinlev-McKee. J. S.. Actu Chem. Scund: 15 (1961) I 4. Silverstein. E.. and Bover, P. D., J. Biol. Chem. 239 I (1964) 398: 5. Dalziel, K., and Dickinson, F. M., Biochem. J. 1 (1966) Theorell, H., and iner, A. D., Arch. Biochem. Biophys. 83 (1959) Theorell, H., and McKinley-McKee, J. S., Actu Chem. Scand. 15 (1961) Theorell, H., and McKinley-McKee, J. S., Acta Chem. Scand. 15 (1961) Yonetani, T., Biochem (1963) Yonetani, T., and Theorell, H., Arch. Biochem. Biophys. 16 (1964) Plane, R. A., and Theorell, H., Actu Chem. Scand. 15 (1961) Kaplan,N. O., and Ciotti,M. M., J. Biol. Chcm. 221 (1954) Kaplan, N. O., Ciotti, M. M., and Stolzenbach, F. E., J. Biol. Chem. 221 (1956) Kaplan, N. O., Ciotti, M. M., and Stolzenbacb, F. E., Arch. Biochem. Biophys. 69 (1957) Shifrin, S., Kaplan, N. O., and Ciotti, M. M., J. Biol. Chem. 234 (1959) Dalziel, K., Biochem. J. 8 (196) Dalziel, K., Actu Chem. Scund. 11 (1957) Dalziel, K., J. Biol. Chem. 238 (1963) Rafter, G.., and Colowick, S. P., Methods i?a Enzymology (edited by S. P. Colowick and N.. Kaplan), Academic Press, New York 1957, Vol. 111, p Dalziel, K., Actu Chem. Scand. 11 (1957) 176. J. D. Shores present address: Department of Biochemistry and Molecular Biology Henry Ford Hospital, Detroit, Mich. 4822, U.S.A. H. Theorell Bioliemiska Avdelningen, Medicinska Nobelinstitutet SolnavLgen 1, Stockholm 6, Sweden