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The Binding of NAD+ and NADH to Glyceraldehydephosphate Dehydrogenase
The Binding of NAD+ and NADH to Glyceraldehydephosphate Dehydrogenase E. C. SLATER, J. J. M. DE VIJLDER,
AND
W. BOERS
Laboratory of Biochemistry, B.C.P. Jansen Institute, University of Amsterdam, Amsterdmm, The Netherlands
I. Introduction . . . 11. Muscle Enzymes . . A. Binding of NAD+ . B. Binding of NADH . C. Mechanism of Action 111. Yeast Enzyme . . . References . . .
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315 316 316 323 323 325 326
I. INTRODUCTION Glyceraldehydephosphate dehydrogenase [~-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating) , EC 1.2.1.121 catalyzes the reaction given in Eq. (1). RCHO
+ NAD+ + Pi
R-COO
N
P
+ NADH + H+
(1)
The physiological substrate is D-glyceraldehyde 3-phosphate, but the enzyme reacts also with other aldehydes (glyceraldehyde, acetaldehyde, and propionaldehyde), although much more slowly. Arsenate can replace phosphate (Needham and Pillai, 1937). In this case the product, an acyl arsenate, hydrolyzes spontaneously so that the sum reaction is RCHO
+ NAD+ + OH- + R*COOH + NADH
(2)
Early studies of its mechanism of action were carried out with enzyme crystallized from yeast (Warburg and Christian, 1939) and rabbit muscle (Cori et al., 1948). More recently, the enzyme isolated from lobster-tail muscle has been used, since the crystals obtained from this source are more suitable for structure determinations by X-ray analysis (Watson and Banaszak, 1964). Only minor differences in properties between the two muscle enzymes have been observed, but the yeast enzyme differs considerably from these two. In all three cases, the enzyme is a tetramer (Harris and Perham, 1965; Harrington and Karr, 1965), composed of four identical subunits each containing 331333 amino acids. The amino acid sequence is known for the lobster-muscle and pig-muscle enzymes (Harris and Perham, 1968). 316
316
E. C. SLATER, J.
J.
M. DE VIJLDER, AND W. BOERS
X-ray analysis has revealed that the lobster-muscle enzyme has at least one 2-fold axis of symmetry (Watson and Banaszak, 1964).
11. MUSCLE ENZYMES A. BINDINGOF NAD+ It was already clear from the early studies that the rabbit-muscle enzyme binds NAD' much more firmly than other dehydrogenases, since the crystalline enzyme contains bound NAD' (Taylor et al., 1948). However, it does not bind NAD' as firmly as activated charcoal, which completely removes NAD' from the enzyme (Velick, 1953). It was early recognized that glyceraldehydephosphate dehydrogenase is the site of the inhibitory action of iodoacetate on glycolysis (Lundsgaard, 1930), and a cysteine side chain was implicated in this inhibition. Although each subunit contains from 2 (yeast) (Harris and Perham, 1963) to 5 (lobster) (Davidson et al., 1967) cysteine residues, only one of these in the native enzyme reacts with iodoacetate, and the rate of this reaction is increased by NAD' (Racker and Krimsky, 1952a). Interestingly, the addition of one molecule of NAD' per tetramer is sufficient maximally to activate the single cysteine residue in all four subunits (Conway and Koshland, 1968). When the enzyme is denatured by urea, all the thiol groups react with iodoacetic acid (Harris and Perham, 1965). Binding of NAD' to charcoal-treated engyme causes the appearance of a broad absorption band with maximum a t 360 nm, and the formation of the band is inhibited by iodoacetate or acetyl phosphate (Racker and Krimsky, 1952a,b; Velick, 1953). Since acetyl phosphate acetylates the same thiol group as iodoacetate, it is clear that this group is concerned, either directly or indirectly, with the formation of the 360-nm band. Indeed, it was thought at one time that a covalent bond is formed between a carbon atom in the nicotinamide ring and the sulfur atom (Eq. 3) and that this bond is split by "aldehydolysis" with the formation of a thiol ester (Eq. 4), followed finally by phosphorolysis (or arsenolysis) of the thiol ester (Eq. 5) (Racker and Krimsky, 1952a,b).
+ +
Sum:
+ +
G S H N A D + e E-S-NAD H+ E--S--NAD RCHO E--5-CO.R NADH I+S-CO*R Pi E-SH RCOOP
(3) (4)
;t
(1)
+ R*CHO+ NAD+ + Pi
+ R.COOP + NADH
(5)
The suggestion that an acylated enzyme-a thiol ester-is an intermediate in the enzyme reaction is now well established (Krimsky and Racker, 1955; Koeppe et al., 1956). It appears unlikely, however, that it
BINDING OF NAD’ AND NADH TO DEHYDELOOENASE
317
is formed by “aldehydolysis” of a carbon-sulfur bond. Kmower (1956) pointed out that the 360-nm band has the characteristics of a chargetransfer complex between an electron donor in the enzyme and the pyridine ring. The electron donor is probably the active thiol group, although the indole group of tryptophan has also been suggested (Cilento and Tedeschi, 1961; see also Boross and Cseke, 1966). The binding constants of NAD+ to each of the four subunits in the muscle enzymes have been determined by Conway and Koshland (1968) by equilibrium dialysis, and by De Vijlder and Slater (1968) and De Vijlder et al. (1969a), using both ultracentrifugation and equilibrium dialysis to separate free NAD’ from that bound to the enzyme. Figure 1 shows the results, given as a Scatchard plot, of the measurements with the lobster enzyme. The curved line, convex to the abscissa, shows that the binding constants become successively less as more N A P becomes bound to the enzyme, i.e., there is negative cooperativity. The inset shows that a straight-line Scatchard plot is obtained when the binding of the fourth molecule is plotted on the assumption that the first three sites are completely occupied before NAD+ is bound to the fourth site. Table I summarizes all the results obtained with the rabbit, lobster, and yeast enzymes. Velick et al. (1970) have shown that the binding constants of
0
1.o
2o
r
3.0
4.0
Fro. 1. Scatchard plot of binding of NAD’ to lobster-muscle enzyme, measured by equilibrium dialysis at 4°C. Inset: Scatchard plot of fourth molecule, calculated on the assumption that the first three sites are completely occupied before NAD’ is bound to the fourth site. r=number of molecules N A P bound per molecule enzyme.
TABLE I DISSOCIATION CONSTANTS OF NAD+ BOUNDm GLYCEEALDEEYDEPEOSPEATE DEEYDEOQENASE
E -
Dissociation constants (M)
p
Rabbit
Reaction
+ NAD+ E N A D + NAD+ G= E(NAD)r ENAD),. + NAD+ eE(NAD)s E ( N A D ) a + NAD+ eE(NAD)r E ENAD
De Vijlder and Slater (1968). At 2625°C. Conway and Koshland (1968). De Vijlder et al. (1969a). 8 Koshland et al. (1970). f At 4°C. a
TJltracentrifugationa.b <5 x <5 x 4 X 3.5 x
10-8 10-6 1 F 106
m P rn
0
Dialysi8c.f
2 . 6 X 106
h b s t e r d (didysisf)
YeaEte (dialyw)
<5 X l P <5 x 106 X lo-’ 1.3 X
5 . 5 x 10-5 4.6 X 1 P 4 . 2 X 1C6 1 . 1 x lo-*
?
-
2 4
“i 9
2 9 W
P
BINDING OF NAD' AND NADH TO DEHYDBOGENASE
319
the first two molecules of NAD' with the rabbit-muscle enzyme, measured by the quenching by added NAD' of the fluorescence of the apoprotein, decrease with increasing temperature, while that of the third is little temperature dependent. The net result is that the differences between the first three molecules disappear a t 36". De Vijlder and Slater (1967, 1968) and D e Vijlder et al. (1969a) showed that only the first three molecules of NAD+ bound to the enzyme contribute to the 360-nm band. This is illustrated for the lobster-muscle enzyme in Fig. 2. The first two molecules of NAD+ are stoichiometrically bound to the enzyme and bring about an equal increase in A36On,n. The departure from the straight line after 2 molecules of NAD+ are added (Fig. 2A) is due to the fact that the third molecule dissociates from the complex with a significant dissociation constant. When A 3 6 0 nm is plotted against enzyme-bound NAD, calculated from that added and the value of 6 x M for the dissociation constant of the third molecule (see Table I ) , it may be seen that this molecule of NAD+ contributes to the 360-nm band to the same extent as the first two (Fig. ZB).The fourth molecule, on the other hand, contributes little, if anything, t o this band. The slightly higher values of A 3 6 0 nm obtained with a large excess of NAD+ is probably due to contact charge-transfer interaction (Kosower, 1960). The 360-nm band is optically active, giving a very broad positive circular dichroism band, centered a t 350 nm, with a molecular elipticity of 18,000 per mole of enzyme (De Vijlder and Hsrmsen, 1969). When changes in the molecular elipticity a t 350 nm of the Moffit-Yang parameter b, are plotted against the amount of NAD+ added to the enzyme, curves similar to those of Fig. 2 are obtained (De Vijlder and Harmsen, 1969). Thus, each of the first three molecules of N A P bound to the enzyme contribute equally to the circular dichroism, and the fourth molecule does not. All the effects of NAD+ addition on the circular dichroism spectrum occur in the region where NAD+ itself gives positive bands, and no changes are observed on the positive circular dichroism band at 299 nm, given by the apoenzyme. The changes observed may thus be ascribed to extrinsic effects of the NAD+ and do not reflect overall changes in the protein conformation. The fourth molecule also differs from the other three in having no effect on the fluorescence of the protein (the first three molecules quench the fluorescence of tryptophan in the molecule; Velick, 1958; Velick et al., 1970) and differs from the third in having no effect on the viscosity of a solution of the protein (the third molecule increases the viscosity; Conway and Koshland, 1968). Of particular interest is the finding of Velick et al. (1970) that binding of the first three molecules of NAD' to the rabbit-muscle enzyme is an exothermic reaction, and that AH is the same
320
E. C. SLATER, J. J . M. DE VIJLDER, AND W. BOERS
moles NAD* added I mole enzyme
A 360 nm
FIQ.2. Titration at 360 nm with N A D of charcoal-treated glyceraldehydephosphate dehydrogenase (28.3 g M ) isolated from lobster muscle. The enzyme waa dissolved in 100 mM Tris-HCl buffer (pH 8.2) containing 5 mM EDTA. Temperature, 23°C. (A) As,., as a function of added NAD'; (B) Amn, as a function of bound
NAD'.
for all three reactions (16 kcal/mole), whereas it is zero for the fourth molecule. From these data and the values for AGO calculated from the binding of NAD+to the enzyme, the change of entropy may be calculated for each of the four reactions. For this calculation, Velick et al. (1970) used binding constants for the first three molecules calculated from the effect of NAD+ at 2 5 O on the protein fluorescence, assuming that each
BINDING OF NAD' AND NADH TO DEHYDBOGENASE
321
molecule of NAD+ has the identical effect. These values (1.7 )( lo-' M , 3.4 X 10-'M and 1.9 X 10-EM) differ somewhat from those found by ultracentrifugation or equilibrium dialysis (Table I). The value used for the binding constant for the fourth molecule was that reported by D e Vijlder and Slater (1968) by ultracentrifugation. I n several respects, then, the binding of NAD+ to the fourth subunit differs from that to the first three. The first molecule also differs in some respects from the second and third. This was shown by De Vijlder and Slater (1967, 1968) with the rabbit enzyme for the rate of reaction between the enzyme and NAD+, as followed in the stopped-flow apparatus by the rate of increase of ASeonm.With less than 1 mole of NAD' per mole of enzyme, an almost maximal increase of the absorbance is reached in 3-5 msec, the mixing time of the instrument. On addition of more than 1 mole of NAD', rapid and slow phases are observed. If the enzyme was previously treated with 1 mole of NAD+ per mole of enzyme, the slow phase on reaction with an additional 1 mole NAD+ was more rapid than when 2 moles of NAD+ were added to NAD+-free enzyme. This may indicate that the first NAD' molecule prepares the binding site for the second molecule. Similarly, the third molecule was bound more rapidly to enzyme already containing two molecules. Strangely, the lobster enzyme differs in this respect from the rabbit muscle. Even st lo", with amounts of NAD+ between 0.7 and 9.4 moles of N D per mole of enzyme, the absorbance change was completed within 3-5 msec of mixing the enzyme with the N A P (De Vijlder et al., 1969a). This corresponds to a second-order rate constant of more than lozoM-lsec-l. These experiments give, of course, no information on the rate of binding of the fourth molecule. The first molecule of NAD+ also activates the reactive -SH group in all four subunits (Conway and Koshland, 1968). The activation involves a lowering of the p R so that, a t neutral pH, these groups become ionized (Stockell, 1959). Finally, as already mentioned, the third molecule differs from the first two in having a lower binding constant a t lower temperatures (Conway and Koshland, 1968; De Vijlder and Slater, 1968; D e Vijlder et al., 1969a) and in the effect of temperature on this binding constant. Thus, differences have been detected at all four binding sites. At physiological temperatures for the rabbit, however, the differences between the fourth site and the other three are quantitatively much more important. It is clear that binding of NAD+ to one subunit invokes asymmetric conformation changes in other subunits. This is not easily explained on the basis of the limiting model for allostery worked out in detail by Monod et al. (1965) and would seem to require a sequential model of the
322
E. C. SLATER, J. J . M. DE VIJLDER, AND W. BOERS
type proposed by Conway and Koshland (1968). Indeed, it scems necessary to invoke four conformations of subunits in order to explain the results, viz. (i) R , in which the -SH group in the active center is not activated (high pR). (ii) S, in which this -SH group is activated (low pK) . (iii) T, a variant of S in which the conformation is changed in such a way that the subunit can combine with NAD+. (iv) U, a variant of T, induced by combination with NAD+, in which the -S- group is favorably placed with respect to the pyridine ring so that a charge-transfer complex may be formed. The four reactions may then be written, for the rabbit-muscle enzyme AGO
(4
+ + +
R( N S SyT*UN SrT*UN N S*T*(UN)r (c) S*T.(UN)s N S T*(UN)a (4 T*(UN)a N TN*(UN)a
(b)
+
(kcal/mole) -9 -9 -8 -6
AH (kcal/mole)
- 16 - 16 -16
0
A&
(entropy unit)
-23
-24
-27 +21
On the basis of this formulation we may draw the following conclusions: 1. The binding of N A P t o T, the conformation ready to receive it (Reaction d), is entropy driven, perhaps as the result of hydrophobic interactions between the protein and the NAD' molecule (Velick et al., 1970). 2. The NAD-induced conformation change S + T + U (Reactions b
and c ) are strongly exothermic. 3. Since, although Reaction a includes the conformation changes 4 R + 4 S and S+ T in addition to the S + T + U also involved in Reactions b and c, the thermodynamic parameters are similar to those for Reactions b and c, it may be concluded that the strongly exothermic conformation change is T + U. I n summary, the binding of NAD+ to the muscle enzyme is dominated thermodynamically by an entropy-driven binding reaction, followed by a strongly exothermic conformation change in three of the subunits. The formation of the charge-transfer complex can contribute to only a minor extent to this conformation change, since the -AG, for the formation of such complexes is usually only about 2-3 kcal/mole (Kosower, 1966). NAD+ is also necessary for the reduction of acyl phosphate by NADH, catalyzed by the enzyme (the reverse of Eq. 1) (Hilvers and Weenen, 1962; Hilvers et al., 1964; D e Vijlder et al., 1969b). and for transfer reactions catalyzed by the enzyme, such as arsenolysis of acyl phosphate (Harting and Velick, 1964). Maximal activity is found with 3 moles of
BINDING OF NAD' AND NADH TO DEHYDROGENASE
323
NAD' bound to the enzyme, indicating that the conformation T.(UN)s is the catalytically active form in these reactions. NAD+ in excess of 3 moles per mole of enzyme inhibits the oxidation of NADH by acyl phosphate, competitive with respect to NADH. The inhibition constant, 45 p H , is close to the dissociation constant of the fourth site (35 &) . Thus, we may write T'(UN)a + NADH T-NADH*(UN)a (6) T*(UN)s+ NAD+ F! T-NAD+*(UN)a (7) where Eq. (6) describes a reaction involved in the oxidation of NADH catalyzed by the enzyme, and Eq. (7) an inhibitory reaction. B. BINDINGOF NADH The binding of NADH to the enzyme has been studied by determining the quenching of NADH fluorescence on binding to the enzyme (Velick, 1953, 1958) and by ultrafiltration (Boers, 1970). Preliminary measurements by the latter technique indicate that the binding constants of the four molecules of NADH are similar to those of the corresponding molecules of NAD+.
C. MECHANISM OF ACTION The rate of oxidation of NADH by acetyl phosphate, in the presence of high concentrations of enzyme, is proportional to the concentration of NADH until 1 mole NADH per mole of enzyme is added. In an experiment described by De Vijlder et al., 196913), the first-order reaction constant with respect to NADH, in the presence of 3 moles of NAD' per mole of enzyme, was 0.19 min-l. Between 1 and 6 moles NADH per mole of enzyme, the first-order constant declined to 0.05 min-l. A similar result was obtained with 1 mole NAD' per mole enzyme. Thus, the first molecule of NADH bound to the enzyme is oxidized more rapidly than subsequent molecules. In contrast, the fourth molecule of NAD' bound is reduced more than twice as rapidly by glyceraldehyde as the other three (De Vijlder et al., 1969b).* The K,,, for this fourth molecule is the same as that for the *The actual values for the catalytic-center activity calculated by De Vijlder et al. (1969b) were 0.014 sec-' for the first three sites and 0.035 sec-' for the fourth. Closely similar results have also been obtained for the lobster enzyme. Teipel and Koshland (1970) have recently calculated a value of 0.78 min" (i.e., 0.013 sec-') for the first three sites in the rabbit enzyme, in excellent agreement with the findings of De Vijlder et al. (196913). The value calculated for the fourth site by Teipel and Koshland (1970) is, however, much less than that of De Vijlder et al. (1969b).The difference appears to lie in the method of calculation, for when the method of De Vijlder et al. was applied to the data of Teipel and Koshland, a value of 0.024 wc-' was obcalculated in this way, was also close tained. The K, for the fourth site (33 to that found by De Vijlder et al. (1969a,b) (17 pM for rabbit, 24 for lobster).
a),
324
E. C. SLATER, J. J. M. DE VIJLDER, AND W. BOERS
overall reaction catalyzed by low concentrations of enzyme, and is also the same as the dissociation constant of the fourth site. These findings and the fact that NAD+ and NADH compete for the fourth site (see above) when the amount of NAD+ exceeds 3 molecules per molecule enzyme suggest that the catalytically most active form of enzyme in both glycolysis (Eq. 1 from left to right) and glucogenesis (Eq. 1 from right to left) is the enzyme with 3 of the 4 subunits occupied by NAD+, and in the conformation that we have written T - ( U N ) , . The catalysis takes place on the fourth subunit. I n the following mechanism T. (UN)3 is written E - S- where S- represents the active thiol group that has formed no charge-transfer complex with NAD'. According to this mechanism, the catalytically active enzyme is a S/ + NAD+ i=E
E-S-
\
NAD+
S-
RCHO
/
+E
e (RCH0)-E
\
NAD+ S-
/
(RCH0)-E
\ NAD+ /
\
S-
NAD+
S4OR
/
+ H+
e E
\
NADH
SGOR
E
\ NADH
F? E-S-COR
SGOR
or
/
/
E
S-
+PiF?E
\
NADH E-S-COR
+ NADH
+ Pi
/
+ R-COOP
\
NADH E-SRCOOP
+
S-
/
E
or
Sum: RCHO
\
eG S -
+ NADH
NADH
+ N U + + Pi e R-COOP + NADH + H+
BINDING OF NAD' AND NADH TO DEHYDBOGENASE
325
conjugated protein with 3 firmly bound prosthetic groups per tetramer. The charge-transfer complex plays no direct role in the enzyme-catalyzed reaction, The fourth NAD+ molecule, which does not form a chargetransfer complex, is a substrate. It cannot be excluded, however, that enzyme molecules containing fewer than 3 molecules of bound NAD+ also play a minor role in the catalysis. The hydride transfer (Eq.iii) may be visualized as in Fig. 3. Experiments with [ l-SH] glyceraldehyde phosphate have shown that, with this enzyme, a direct hydrogen transfer from the aldehyde to the nicotinamide takes place (Allison et al., 1969), in contrast to alcohol, lactate, and malate dehydrogenases, where hydrogen transfer between substrate and nicotinamide occurs via a tryptophan in the protein (Chan and Schellenberg, 1968). R
R
\
NO6-
B\ so H
Enzyme
\C/p
I
NAD+
Enzyme
Enzyme
FIG.3. Proposed mechanism of hydride transfer to NAD bound to fourth site of glyceraldehydephosphate dehydrogenase.
111. YEASTENZYME Remarkable differences between the behavior of the muscle and yeast enzymes have been reported despite the fact that the primary structures are sufficiently similar to allow the formation of hybrid tetramers (Kirschner and Schuster, 1970). These differences are: 1. The binding constants are so much less (Table I) that the yeast enzyme as usually prepared contains little NAD+ (Warburg and Christian, 1939). 2. A positive cooperativity is observed in the binding of successive molecules of NAD+, especially at higher temperatures. There is a difference of opinion between Koshland et al., (1970) (see Table I ) , who has reported a mixture of positive cooperativity (between first and second sites) and negative cooperativity (between second and third, and third and fourth) and Kirschner and co-workers (1966; Kirschner, 1968; Kirschner and Schuster, 1970), who consider that only positive cooperativity is present.
326
E. C. SLATER, J. J . M. DE VIJLDER, AND W. BOERS
3. According to Kirschner and Schuster (1970) and Chance and Harting Park (1967), all four NAD+molecules contribute equally to the 360nm band. However, if the low value of Koshland et al. (1970) for the binding constant of the fourth site is correct, the fourth site could scarcely have been occupied in the experiments of Kirschner and Schuster (1970) and Chance and Harting Park (1967). Kirschner and co-workers interpret the results in terms of the allosteric model of Monod et al. (1965), which may be written
+ N ekDR r R N &*RN + N e Rr(RN)r Rn.(RN), + N * R.(RN)s R*(RN)a+ N (RN). kn
+ + +
TrTN N Tn.(TN)* 'h(TN), N e T*(TN)a T*(TN), N e (TN),
Kinetic studies by rapid-mixing and temperature-jump techniques yielded the following values at pH 9.0 and 20" (Kirschner and Schuster, 1970) :
Ico kr
1.2 see-1 0.05 sec-' 5.3 X 106M-' 8ec-I
]cD.
1.25 X 10' sec-'
k4
La KR
3
=
24 3.0 X 10-sM
= intrinsic dissociation constant of R form with NAD+; K T = intrinsic dissociation constant of T form with NAD'. The T state is enzymatically inactive. The transition from the R to the T state is accompanied by an increase in anisotropy and a volume contraction of 7%. Indeed the T state can be regarded as a reversible denatured state of the enzyme. K R
REFERENCES Allison, W. S., Connors, M. J., and Parker, D.J. (1969). Biochem. Biophys. Res. Commun. 34, 503. Boers, W. (1970). Unpublished data. Boross, L., and Cseke, E. (1966). Acta Biochim. Biophys. 2,47. Chan, T. L., and Schellenberg,K. A. (1968). J . B i d . Chem. 243,6284. Chance, B., and Harting Park, J. (1967). J . Biol. Chem. 242, 5093. Cilento, G.,and Tedeschi, P. (1961). J . Biol. Chem. 236,907. Conway, A.,and Koshland, D.E.,Jr., (1968). Biochemistry 7,4011. Cori, G.T., Slein, M. W., and Cori, C. F. (1948). J. BWZ.Chem. 173,605.
BINDING OF NAD’ AND NADH TO DEHYDBOGENASE
327
Davidson, B. E., Sajgb, M., Noller, H. F., and Harris, J. I. (1967). Nature (London) 216, 1181. De Vijlder, J. J. M., and Harmsen, B. J. M. (1969). Biochim. Biophys. Acta 178, 434. De Vijlder, J. J. M., and Slater, E. C. (1967). Biochim. BWphys. Acta 132, 207. De Vijlder, J. J. M., and Slater, E. C. (1968). Biochim. Biophys. Acta 167, 23. De Vijlder, J. J. M., Boers, W., and Slater, E. C. (1969a). Bwchim. Biophys. Acta 191, 214. De Vijlder, J. J. M., Hilvers, A. G., Van Lis, J. M. J., and Slater, E. C. (196913). Biochim. Bwphys. Acta 191,221. Harrington, W. F., and Karr, G. M. (1965). J. Mol. B i d . 13,885. Harris, J. I., and Perham, R. N. (1963). Bwchem. J . 89,60P. Harris, J. I., and Perham, R. N. (1965). J. MoZ. BWZ. 13,876. Harris, J. I., and Perham, R. N. (1968). Nature (London) 219,1025. Harting, J., and Velick, S. F. (1954). J. BWl. Chem. 207,857. Hilvers, A. G., and Weenen, J. H. M. (1962). Bwchkm. Biophys. Acta 58, 380. Hilvers, A. G., Van Dam, K., and Slater, E. C. (1964). Biochim. Bwphys. Acta 85, 206.
Kirschner, K. (1968). In “Regulation of Enzyme Activity and Allosteric Interactions” (E. Kvamme and A. Pihl, eds.), p. 39. Academic Press, New York. Kirschner, K., and Schuster, I. (1970). In “Pyridine Nucleotide-Dependent Dehydrogenases” (H. Sund, ed.), p. 217. Springer, Berlin. Kirschner, K., Eigen, M., Bittman, R., and Voigt, B. (1966). Proc. Nut. Acad. Sci. U.8.56, 1661. Koeppe, 0 . J., Boyer, P. D., and Stulberg, M. P. (1956). J. BWZ. Chem. 219, 569. Koshland, D. E., Jr., Cook, R. A., and Cornish-Bowden, A. (1970). I n “Pyridine Nucleotide-Dependent Dehydrogenaaes” (H. Sund, ed.), p. 199. Springer, Berlin. Kosower, E. M. (1956). J. Amer. Chem. SOC.78,3497. Kosower, E. M. (1960). In “The Enzymes” (P. D. Boyer, H. Lardy, and K. Myrbiick, eds.), 2nd rev. ed., Vol. 3, p. 171. Academic Press, New York. Kosower, E. M. (1966). In “Flavins and Flavoproteins” (E. C. Slater, ed.), B.B.A. Library, Vol. 8, p. 1. Elsevier, Amsterdam. Krimsky, I., and Racker, E. (1955). Science 122,319. Lundsgaard, E. (1930). Biochem. 2.227, 51. Monod, J., Wyman, J., and Changeux, J. P. (1965). J. Mol. Biol. 1 2 , s . Needham, D. M., and Pillai, R. K. (1937). Biochem. J. 31, 1837. Racker, E., and Krimsky, I. (1952a). Nature (London) 169, 1043. Racker, E., and Krimsky, I. (1952b). J. BWZ. Chem. 198,731. Stockell, A. (1959). J. B i d . Chem. 234, 1286. Taylor, J. F., Velick, S. F., Cori, G. T.,‘Cori, C. F., and Slein, M. W. (1948). J. B i d . Chem. 173, 619. Teipel, J., and Koshland, D. E., Jr. (1970). Biochim. Bwphys. Actu 198, 183. Velick, S. F. (1953). J. BWZ. Chem. 203, 563. Velick, S. F. (1958). J. B i d . Chem. 233, 1455. Velick, S. F., Baggott, J. P., and Sturtevant, J. M. (1970). In “Pyridine NucleotideDependent Dehydrogenases” (H. Sund, ed.), p. 229. Springer, Berlin. Warburg, 0.. and Christian, W. (1939). Bbchem. 2.303,40. Watson, H. C., and Banaszak, L. J. (1964). Nature (London) 204,918.
AND
W. BOERS
Laboratory of Biochemistry, B.C.P. Jansen Institute, University of Amsterdam, Amsterdmm, The Netherlands
I. Introduction . . . 11. Muscle Enzymes . . A. Binding of NAD+ . B. Binding of NADH . C. Mechanism of Action 111. Yeast Enzyme . . . References . . .
. . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . .
. . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
315 316 316 323 323 325 326
I. INTRODUCTION Glyceraldehydephosphate dehydrogenase [~-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating) , EC 1.2.1.121 catalyzes the reaction given in Eq. (1). RCHO
+ NAD+ + Pi
R-COO
N
P
+ NADH + H+
(1)
The physiological substrate is D-glyceraldehyde 3-phosphate, but the enzyme reacts also with other aldehydes (glyceraldehyde, acetaldehyde, and propionaldehyde), although much more slowly. Arsenate can replace phosphate (Needham and Pillai, 1937). In this case the product, an acyl arsenate, hydrolyzes spontaneously so that the sum reaction is RCHO
+ NAD+ + OH- + R*COOH + NADH
(2)
Early studies of its mechanism of action were carried out with enzyme crystallized from yeast (Warburg and Christian, 1939) and rabbit muscle (Cori et al., 1948). More recently, the enzyme isolated from lobster-tail muscle has been used, since the crystals obtained from this source are more suitable for structure determinations by X-ray analysis (Watson and Banaszak, 1964). Only minor differences in properties between the two muscle enzymes have been observed, but the yeast enzyme differs considerably from these two. In all three cases, the enzyme is a tetramer (Harris and Perham, 1965; Harrington and Karr, 1965), composed of four identical subunits each containing 331333 amino acids. The amino acid sequence is known for the lobster-muscle and pig-muscle enzymes (Harris and Perham, 1968). 316
316
E. C. SLATER, J.
J.
M. DE VIJLDER, AND W. BOERS
X-ray analysis has revealed that the lobster-muscle enzyme has at least one 2-fold axis of symmetry (Watson and Banaszak, 1964).
11. MUSCLE ENZYMES A. BINDINGOF NAD+ It was already clear from the early studies that the rabbit-muscle enzyme binds NAD' much more firmly than other dehydrogenases, since the crystalline enzyme contains bound NAD' (Taylor et al., 1948). However, it does not bind NAD' as firmly as activated charcoal, which completely removes NAD' from the enzyme (Velick, 1953). It was early recognized that glyceraldehydephosphate dehydrogenase is the site of the inhibitory action of iodoacetate on glycolysis (Lundsgaard, 1930), and a cysteine side chain was implicated in this inhibition. Although each subunit contains from 2 (yeast) (Harris and Perham, 1963) to 5 (lobster) (Davidson et al., 1967) cysteine residues, only one of these in the native enzyme reacts with iodoacetate, and the rate of this reaction is increased by NAD' (Racker and Krimsky, 1952a). Interestingly, the addition of one molecule of NAD' per tetramer is sufficient maximally to activate the single cysteine residue in all four subunits (Conway and Koshland, 1968). When the enzyme is denatured by urea, all the thiol groups react with iodoacetic acid (Harris and Perham, 1965). Binding of NAD' to charcoal-treated engyme causes the appearance of a broad absorption band with maximum a t 360 nm, and the formation of the band is inhibited by iodoacetate or acetyl phosphate (Racker and Krimsky, 1952a,b; Velick, 1953). Since acetyl phosphate acetylates the same thiol group as iodoacetate, it is clear that this group is concerned, either directly or indirectly, with the formation of the 360-nm band. Indeed, it was thought at one time that a covalent bond is formed between a carbon atom in the nicotinamide ring and the sulfur atom (Eq. 3) and that this bond is split by "aldehydolysis" with the formation of a thiol ester (Eq. 4), followed finally by phosphorolysis (or arsenolysis) of the thiol ester (Eq. 5) (Racker and Krimsky, 1952a,b).
+ +
Sum:
+ +
G S H N A D + e E-S-NAD H+ E--S--NAD RCHO E--5-CO.R NADH I+S-CO*R Pi E-SH RCOOP
(3) (4)
;t
(1)
+ R*CHO+ NAD+ + Pi
+ R.COOP + NADH
(5)
The suggestion that an acylated enzyme-a thiol ester-is an intermediate in the enzyme reaction is now well established (Krimsky and Racker, 1955; Koeppe et al., 1956). It appears unlikely, however, that it
BINDING OF NAD’ AND NADH TO DEHYDELOOENASE
317
is formed by “aldehydolysis” of a carbon-sulfur bond. Kmower (1956) pointed out that the 360-nm band has the characteristics of a chargetransfer complex between an electron donor in the enzyme and the pyridine ring. The electron donor is probably the active thiol group, although the indole group of tryptophan has also been suggested (Cilento and Tedeschi, 1961; see also Boross and Cseke, 1966). The binding constants of NAD+ to each of the four subunits in the muscle enzymes have been determined by Conway and Koshland (1968) by equilibrium dialysis, and by De Vijlder and Slater (1968) and De Vijlder et al. (1969a), using both ultracentrifugation and equilibrium dialysis to separate free NAD’ from that bound to the enzyme. Figure 1 shows the results, given as a Scatchard plot, of the measurements with the lobster enzyme. The curved line, convex to the abscissa, shows that the binding constants become successively less as more N A P becomes bound to the enzyme, i.e., there is negative cooperativity. The inset shows that a straight-line Scatchard plot is obtained when the binding of the fourth molecule is plotted on the assumption that the first three sites are completely occupied before NAD+ is bound to the fourth site. Table I summarizes all the results obtained with the rabbit, lobster, and yeast enzymes. Velick et al. (1970) have shown that the binding constants of
0
1.o
2o
r
3.0
4.0
Fro. 1. Scatchard plot of binding of NAD’ to lobster-muscle enzyme, measured by equilibrium dialysis at 4°C. Inset: Scatchard plot of fourth molecule, calculated on the assumption that the first three sites are completely occupied before NAD’ is bound to the fourth site. r=number of molecules N A P bound per molecule enzyme.
TABLE I DISSOCIATION CONSTANTS OF NAD+ BOUNDm GLYCEEALDEEYDEPEOSPEATE DEEYDEOQENASE
E -
Dissociation constants (M)
p
Rabbit
Reaction
+ NAD+ E N A D + NAD+ G= E(NAD)r ENAD),. + NAD+ eE(NAD)s E ( N A D ) a + NAD+ eE(NAD)r E ENAD
De Vijlder and Slater (1968). At 2625°C. Conway and Koshland (1968). De Vijlder et al. (1969a). 8 Koshland et al. (1970). f At 4°C. a
TJltracentrifugationa.b <5 x <5 x 4 X 3.5 x
10-8 10-6 1 F 106
m P rn
0
Dialysi8c.f
2 . 6 X 106
h b s t e r d (didysisf)
YeaEte (dialyw)
<5 X l P <5 x 106 X lo-’ 1.3 X
5 . 5 x 10-5 4.6 X 1 P 4 . 2 X 1C6 1 . 1 x lo-*
?
-
2 4
“i 9
2 9 W
P
BINDING OF NAD' AND NADH TO DEHYDBOGENASE
319
the first two molecules of NAD' with the rabbit-muscle enzyme, measured by the quenching by added NAD' of the fluorescence of the apoprotein, decrease with increasing temperature, while that of the third is little temperature dependent. The net result is that the differences between the first three molecules disappear a t 36". De Vijlder and Slater (1967, 1968) and D e Vijlder et al. (1969a) showed that only the first three molecules of NAD+ bound to the enzyme contribute to the 360-nm band. This is illustrated for the lobster-muscle enzyme in Fig. 2. The first two molecules of NAD+ are stoichiometrically bound to the enzyme and bring about an equal increase in A36On,n. The departure from the straight line after 2 molecules of NAD+ are added (Fig. 2A) is due to the fact that the third molecule dissociates from the complex with a significant dissociation constant. When A 3 6 0 nm is plotted against enzyme-bound NAD, calculated from that added and the value of 6 x M for the dissociation constant of the third molecule (see Table I ) , it may be seen that this molecule of NAD+ contributes to the 360-nm band to the same extent as the first two (Fig. ZB).The fourth molecule, on the other hand, contributes little, if anything, t o this band. The slightly higher values of A 3 6 0 nm obtained with a large excess of NAD+ is probably due to contact charge-transfer interaction (Kosower, 1960). The 360-nm band is optically active, giving a very broad positive circular dichroism band, centered a t 350 nm, with a molecular elipticity of 18,000 per mole of enzyme (De Vijlder and Hsrmsen, 1969). When changes in the molecular elipticity a t 350 nm of the Moffit-Yang parameter b, are plotted against the amount of NAD+ added to the enzyme, curves similar to those of Fig. 2 are obtained (De Vijlder and Harmsen, 1969). Thus, each of the first three molecules of N A P bound to the enzyme contribute equally to the circular dichroism, and the fourth molecule does not. All the effects of NAD+ addition on the circular dichroism spectrum occur in the region where NAD+ itself gives positive bands, and no changes are observed on the positive circular dichroism band at 299 nm, given by the apoenzyme. The changes observed may thus be ascribed to extrinsic effects of the NAD+ and do not reflect overall changes in the protein conformation. The fourth molecule also differs from the other three in having no effect on the fluorescence of the protein (the first three molecules quench the fluorescence of tryptophan in the molecule; Velick, 1958; Velick et al., 1970) and differs from the third in having no effect on the viscosity of a solution of the protein (the third molecule increases the viscosity; Conway and Koshland, 1968). Of particular interest is the finding of Velick et al. (1970) that binding of the first three molecules of NAD' to the rabbit-muscle enzyme is an exothermic reaction, and that AH is the same
320
E. C. SLATER, J. J . M. DE VIJLDER, AND W. BOERS
moles NAD* added I mole enzyme
A 360 nm
FIQ.2. Titration at 360 nm with N A D of charcoal-treated glyceraldehydephosphate dehydrogenase (28.3 g M ) isolated from lobster muscle. The enzyme waa dissolved in 100 mM Tris-HCl buffer (pH 8.2) containing 5 mM EDTA. Temperature, 23°C. (A) As,., as a function of added NAD'; (B) Amn, as a function of bound
NAD'.
for all three reactions (16 kcal/mole), whereas it is zero for the fourth molecule. From these data and the values for AGO calculated from the binding of NAD+to the enzyme, the change of entropy may be calculated for each of the four reactions. For this calculation, Velick et al. (1970) used binding constants for the first three molecules calculated from the effect of NAD+ at 2 5 O on the protein fluorescence, assuming that each
BINDING OF NAD' AND NADH TO DEHYDBOGENASE
321
molecule of NAD+ has the identical effect. These values (1.7 )( lo-' M , 3.4 X 10-'M and 1.9 X 10-EM) differ somewhat from those found by ultracentrifugation or equilibrium dialysis (Table I). The value used for the binding constant for the fourth molecule was that reported by D e Vijlder and Slater (1968) by ultracentrifugation. I n several respects, then, the binding of NAD+ to the fourth subunit differs from that to the first three. The first molecule also differs in some respects from the second and third. This was shown by De Vijlder and Slater (1967, 1968) with the rabbit enzyme for the rate of reaction between the enzyme and NAD+, as followed in the stopped-flow apparatus by the rate of increase of ASeonm.With less than 1 mole of NAD' per mole of enzyme, an almost maximal increase of the absorbance is reached in 3-5 msec, the mixing time of the instrument. On addition of more than 1 mole of NAD', rapid and slow phases are observed. If the enzyme was previously treated with 1 mole of NAD+ per mole of enzyme, the slow phase on reaction with an additional 1 mole NAD+ was more rapid than when 2 moles of NAD+ were added to NAD+-free enzyme. This may indicate that the first NAD' molecule prepares the binding site for the second molecule. Similarly, the third molecule was bound more rapidly to enzyme already containing two molecules. Strangely, the lobster enzyme differs in this respect from the rabbit muscle. Even st lo", with amounts of NAD+ between 0.7 and 9.4 moles of N D per mole of enzyme, the absorbance change was completed within 3-5 msec of mixing the enzyme with the N A P (De Vijlder et al., 1969a). This corresponds to a second-order rate constant of more than lozoM-lsec-l. These experiments give, of course, no information on the rate of binding of the fourth molecule. The first molecule of NAD+ also activates the reactive -SH group in all four subunits (Conway and Koshland, 1968). The activation involves a lowering of the p R so that, a t neutral pH, these groups become ionized (Stockell, 1959). Finally, as already mentioned, the third molecule differs from the first two in having a lower binding constant a t lower temperatures (Conway and Koshland, 1968; De Vijlder and Slater, 1968; D e Vijlder et al., 1969a) and in the effect of temperature on this binding constant. Thus, differences have been detected at all four binding sites. At physiological temperatures for the rabbit, however, the differences between the fourth site and the other three are quantitatively much more important. It is clear that binding of NAD+ to one subunit invokes asymmetric conformation changes in other subunits. This is not easily explained on the basis of the limiting model for allostery worked out in detail by Monod et al. (1965) and would seem to require a sequential model of the
322
E. C. SLATER, J. J . M. DE VIJLDER, AND W. BOERS
type proposed by Conway and Koshland (1968). Indeed, it scems necessary to invoke four conformations of subunits in order to explain the results, viz. (i) R , in which the -SH group in the active center is not activated (high pR). (ii) S, in which this -SH group is activated (low pK) . (iii) T, a variant of S in which the conformation is changed in such a way that the subunit can combine with NAD+. (iv) U, a variant of T, induced by combination with NAD+, in which the -S- group is favorably placed with respect to the pyridine ring so that a charge-transfer complex may be formed. The four reactions may then be written, for the rabbit-muscle enzyme AGO
(4
+ + +
R( N S SyT*UN SrT*UN N S*T*(UN)r (c) S*T.(UN)s N S T*(UN)a (4 T*(UN)a N TN*(UN)a
(b)
+
(kcal/mole) -9 -9 -8 -6
AH (kcal/mole)
- 16 - 16 -16
0
A&
(entropy unit)
-23
-24
-27 +21
On the basis of this formulation we may draw the following conclusions: 1. The binding of N A P t o T, the conformation ready to receive it (Reaction d), is entropy driven, perhaps as the result of hydrophobic interactions between the protein and the NAD' molecule (Velick et al., 1970). 2. The NAD-induced conformation change S + T + U (Reactions b
and c ) are strongly exothermic. 3. Since, although Reaction a includes the conformation changes 4 R + 4 S and S+ T in addition to the S + T + U also involved in Reactions b and c, the thermodynamic parameters are similar to those for Reactions b and c, it may be concluded that the strongly exothermic conformation change is T + U. I n summary, the binding of NAD+ to the muscle enzyme is dominated thermodynamically by an entropy-driven binding reaction, followed by a strongly exothermic conformation change in three of the subunits. The formation of the charge-transfer complex can contribute to only a minor extent to this conformation change, since the -AG, for the formation of such complexes is usually only about 2-3 kcal/mole (Kosower, 1966). NAD+ is also necessary for the reduction of acyl phosphate by NADH, catalyzed by the enzyme (the reverse of Eq. 1) (Hilvers and Weenen, 1962; Hilvers et al., 1964; D e Vijlder et al., 1969b). and for transfer reactions catalyzed by the enzyme, such as arsenolysis of acyl phosphate (Harting and Velick, 1964). Maximal activity is found with 3 moles of
BINDING OF NAD' AND NADH TO DEHYDROGENASE
323
NAD' bound to the enzyme, indicating that the conformation T.(UN)s is the catalytically active form in these reactions. NAD+ in excess of 3 moles per mole of enzyme inhibits the oxidation of NADH by acyl phosphate, competitive with respect to NADH. The inhibition constant, 45 p H , is close to the dissociation constant of the fourth site (35 &) . Thus, we may write T'(UN)a + NADH T-NADH*(UN)a (6) T*(UN)s+ NAD+ F! T-NAD+*(UN)a (7) where Eq. (6) describes a reaction involved in the oxidation of NADH catalyzed by the enzyme, and Eq. (7) an inhibitory reaction. B. BINDINGOF NADH The binding of NADH to the enzyme has been studied by determining the quenching of NADH fluorescence on binding to the enzyme (Velick, 1953, 1958) and by ultrafiltration (Boers, 1970). Preliminary measurements by the latter technique indicate that the binding constants of the four molecules of NADH are similar to those of the corresponding molecules of NAD+.
C. MECHANISM OF ACTION The rate of oxidation of NADH by acetyl phosphate, in the presence of high concentrations of enzyme, is proportional to the concentration of NADH until 1 mole NADH per mole of enzyme is added. In an experiment described by De Vijlder et al., 196913), the first-order reaction constant with respect to NADH, in the presence of 3 moles of NAD' per mole of enzyme, was 0.19 min-l. Between 1 and 6 moles NADH per mole of enzyme, the first-order constant declined to 0.05 min-l. A similar result was obtained with 1 mole NAD' per mole enzyme. Thus, the first molecule of NADH bound to the enzyme is oxidized more rapidly than subsequent molecules. In contrast, the fourth molecule of NAD' bound is reduced more than twice as rapidly by glyceraldehyde as the other three (De Vijlder et al., 1969b).* The K,,, for this fourth molecule is the same as that for the *The actual values for the catalytic-center activity calculated by De Vijlder et al. (1969b) were 0.014 sec-' for the first three sites and 0.035 sec-' for the fourth. Closely similar results have also been obtained for the lobster enzyme. Teipel and Koshland (1970) have recently calculated a value of 0.78 min" (i.e., 0.013 sec-') for the first three sites in the rabbit enzyme, in excellent agreement with the findings of De Vijlder et al. (196913). The value calculated for the fourth site by Teipel and Koshland (1970) is, however, much less than that of De Vijlder et al. (1969b).The difference appears to lie in the method of calculation, for when the method of De Vijlder et al. was applied to the data of Teipel and Koshland, a value of 0.024 wc-' was obcalculated in this way, was also close tained. The K, for the fourth site (33 to that found by De Vijlder et al. (1969a,b) (17 pM for rabbit, 24 for lobster).
a),
324
E. C. SLATER, J. J. M. DE VIJLDER, AND W. BOERS
overall reaction catalyzed by low concentrations of enzyme, and is also the same as the dissociation constant of the fourth site. These findings and the fact that NAD+ and NADH compete for the fourth site (see above) when the amount of NAD+ exceeds 3 molecules per molecule enzyme suggest that the catalytically most active form of enzyme in both glycolysis (Eq. 1 from left to right) and glucogenesis (Eq. 1 from right to left) is the enzyme with 3 of the 4 subunits occupied by NAD+, and in the conformation that we have written T - ( U N ) , . The catalysis takes place on the fourth subunit. I n the following mechanism T. (UN)3 is written E - S- where S- represents the active thiol group that has formed no charge-transfer complex with NAD'. According to this mechanism, the catalytically active enzyme is a S/ + NAD+ i=E
E-S-
\
NAD+
S-
RCHO
/
+E
e (RCH0)-E
\
NAD+ S-
/
(RCH0)-E
\ NAD+ /
\
S-
NAD+
S4OR
/
+ H+
e E
\
NADH
SGOR
E
\ NADH
F? E-S-COR
SGOR
or
/
/
E
S-
+PiF?E
\
NADH E-S-COR
+ NADH
+ Pi
/
+ R-COOP
\
NADH E-SRCOOP
+
S-
/
E
or
Sum: RCHO
\
eG S -
+ NADH
NADH
+ N U + + Pi e R-COOP + NADH + H+
BINDING OF NAD' AND NADH TO DEHYDBOGENASE
325
conjugated protein with 3 firmly bound prosthetic groups per tetramer. The charge-transfer complex plays no direct role in the enzyme-catalyzed reaction, The fourth NAD+ molecule, which does not form a chargetransfer complex, is a substrate. It cannot be excluded, however, that enzyme molecules containing fewer than 3 molecules of bound NAD+ also play a minor role in the catalysis. The hydride transfer (Eq.iii) may be visualized as in Fig. 3. Experiments with [ l-SH] glyceraldehyde phosphate have shown that, with this enzyme, a direct hydrogen transfer from the aldehyde to the nicotinamide takes place (Allison et al., 1969), in contrast to alcohol, lactate, and malate dehydrogenases, where hydrogen transfer between substrate and nicotinamide occurs via a tryptophan in the protein (Chan and Schellenberg, 1968). R
R
\
NO6-
B\ so H
Enzyme
\C/p
I
NAD+
Enzyme
Enzyme
FIG.3. Proposed mechanism of hydride transfer to NAD bound to fourth site of glyceraldehydephosphate dehydrogenase.
111. YEASTENZYME Remarkable differences between the behavior of the muscle and yeast enzymes have been reported despite the fact that the primary structures are sufficiently similar to allow the formation of hybrid tetramers (Kirschner and Schuster, 1970). These differences are: 1. The binding constants are so much less (Table I) that the yeast enzyme as usually prepared contains little NAD+ (Warburg and Christian, 1939). 2. A positive cooperativity is observed in the binding of successive molecules of NAD+, especially at higher temperatures. There is a difference of opinion between Koshland et al., (1970) (see Table I ) , who has reported a mixture of positive cooperativity (between first and second sites) and negative cooperativity (between second and third, and third and fourth) and Kirschner and co-workers (1966; Kirschner, 1968; Kirschner and Schuster, 1970), who consider that only positive cooperativity is present.
326
E. C. SLATER, J. J . M. DE VIJLDER, AND W. BOERS
3. According to Kirschner and Schuster (1970) and Chance and Harting Park (1967), all four NAD+molecules contribute equally to the 360nm band. However, if the low value of Koshland et al. (1970) for the binding constant of the fourth site is correct, the fourth site could scarcely have been occupied in the experiments of Kirschner and Schuster (1970) and Chance and Harting Park (1967). Kirschner and co-workers interpret the results in terms of the allosteric model of Monod et al. (1965), which may be written
+ N ekDR r R N &*RN + N e Rr(RN)r Rn.(RN), + N * R.(RN)s R*(RN)a+ N (RN). kn
+ + +
TrTN N Tn.(TN)* 'h(TN), N e T*(TN)a T*(TN), N e (TN),
Kinetic studies by rapid-mixing and temperature-jump techniques yielded the following values at pH 9.0 and 20" (Kirschner and Schuster, 1970) :
Ico kr
1.2 see-1 0.05 sec-' 5.3 X 106M-' 8ec-I
]cD.
1.25 X 10' sec-'
k4
La KR
3
=
24 3.0 X 10-sM
= intrinsic dissociation constant of R form with NAD+; K T = intrinsic dissociation constant of T form with NAD'. The T state is enzymatically inactive. The transition from the R to the T state is accompanied by an increase in anisotropy and a volume contraction of 7%. Indeed the T state can be regarded as a reversible denatured state of the enzyme. K R
REFERENCES Allison, W. S., Connors, M. J., and Parker, D.J. (1969). Biochem. Biophys. Res. Commun. 34, 503. Boers, W. (1970). Unpublished data. Boross, L., and Cseke, E. (1966). Acta Biochim. Biophys. 2,47. Chan, T. L., and Schellenberg,K. A. (1968). J . B i d . Chem. 243,6284. Chance, B., and Harting Park, J. (1967). J . Biol. Chem. 242, 5093. Cilento, G.,and Tedeschi, P. (1961). J . Biol. Chem. 236,907. Conway, A.,and Koshland, D.E.,Jr., (1968). Biochemistry 7,4011. Cori, G.T., Slein, M. W., and Cori, C. F. (1948). J. BWZ.Chem. 173,605.
BINDING OF NAD’ AND NADH TO DEHYDBOGENASE
327
Davidson, B. E., Sajgb, M., Noller, H. F., and Harris, J. I. (1967). Nature (London) 216, 1181. De Vijlder, J. J. M., and Harmsen, B. J. M. (1969). Biochim. Biophys. Acta 178, 434. De Vijlder, J. J. M., and Slater, E. C. (1967). Biochim. BWphys. Acta 132, 207. De Vijlder, J. J. M., and Slater, E. C. (1968). Biochim. Biophys. Acta 167, 23. De Vijlder, J. J. M., Boers, W., and Slater, E. C. (1969a). Bwchim. Biophys. Acta 191, 214. De Vijlder, J. J. M., Hilvers, A. G., Van Lis, J. M. J., and Slater, E. C. (196913). Biochim. Bwphys. Acta 191,221. Harrington, W. F., and Karr, G. M. (1965). J. Mol. B i d . 13,885. Harris, J. I., and Perham, R. N. (1963). Bwchem. J . 89,60P. Harris, J. I., and Perham, R. N. (1965). J. MoZ. BWZ. 13,876. Harris, J. I., and Perham, R. N. (1968). Nature (London) 219,1025. Harting, J., and Velick, S. F. (1954). J. BWl. Chem. 207,857. Hilvers, A. G., and Weenen, J. H. M. (1962). Bwchkm. Biophys. Acta 58, 380. Hilvers, A. G., Van Dam, K., and Slater, E. C. (1964). Biochim. Bwphys. Acta 85, 206.
Kirschner, K. (1968). In “Regulation of Enzyme Activity and Allosteric Interactions” (E. Kvamme and A. Pihl, eds.), p. 39. Academic Press, New York. Kirschner, K., and Schuster, I. (1970). In “Pyridine Nucleotide-Dependent Dehydrogenases” (H. Sund, ed.), p. 217. Springer, Berlin. Kirschner, K., Eigen, M., Bittman, R., and Voigt, B. (1966). Proc. Nut. Acad. Sci. U.8.56, 1661. Koeppe, 0 . J., Boyer, P. D., and Stulberg, M. P. (1956). J. BWZ. Chem. 219, 569. Koshland, D. E., Jr., Cook, R. A., and Cornish-Bowden, A. (1970). I n “Pyridine Nucleotide-Dependent Dehydrogenaaes” (H. Sund, ed.), p. 199. Springer, Berlin. Kosower, E. M. (1956). J. Amer. Chem. SOC.78,3497. Kosower, E. M. (1960). In “The Enzymes” (P. D. Boyer, H. Lardy, and K. Myrbiick, eds.), 2nd rev. ed., Vol. 3, p. 171. Academic Press, New York. Kosower, E. M. (1966). In “Flavins and Flavoproteins” (E. C. Slater, ed.), B.B.A. Library, Vol. 8, p. 1. Elsevier, Amsterdam. Krimsky, I., and Racker, E. (1955). Science 122,319. Lundsgaard, E. (1930). Biochem. 2.227, 51. Monod, J., Wyman, J., and Changeux, J. P. (1965). J. Mol. Biol. 1 2 , s . Needham, D. M., and Pillai, R. K. (1937). Biochem. J. 31, 1837. Racker, E., and Krimsky, I. (1952a). Nature (London) 169, 1043. Racker, E., and Krimsky, I. (1952b). J. BWZ. Chem. 198,731. Stockell, A. (1959). J. B i d . Chem. 234, 1286. Taylor, J. F., Velick, S. F., Cori, G. T.,‘Cori, C. F., and Slein, M. W. (1948). J. B i d . Chem. 173, 619. Teipel, J., and Koshland, D. E., Jr. (1970). Biochim. Bwphys. Actu 198, 183. Velick, S. F. (1953). J. BWZ. Chem. 203, 563. Velick, S. F. (1958). J. B i d . Chem. 233, 1455. Velick, S. F., Baggott, J. P., and Sturtevant, J. M. (1970). In “Pyridine NucleotideDependent Dehydrogenases” (H. Sund, ed.), p. 229. Springer, Berlin. Warburg, 0.. and Christian, W. (1939). Bbchem. 2.303,40. Watson, H. C., and Banaszak, L. J. (1964). Nature (London) 204,918.