Covalent Binding of 3-Pyridinealdehyde Nicotinamide Adenine Dinucleotide and Substrate to Glyceraldehyde S-Phosphate Dehydrogenase*

Glyceraldehyde 3-phosphate dehydrogenase (D-glyceraldehyde-3-phoshate:nicotinamide adenine dinucleotide oxidoreductase (phosphorylating), EC 1.2.1.12) forms a complex with 3-pyridinealdehyde-NAD which survives precipitation with 7% perchloric acid. The molar ratio bound 3-pyridinealdehyde-NAD to the enzyme is 2.5 to 2.9. Lactate, malate, and alcohol dehydrogenases do not form acid-precipitable complexes with 3-pyridinealdehyde-NAD. 3-Pyridinealdehyde-deamino-NAD or glyceraldehyde 3-phosphate also forms an acid-stable complex with glyceraldehyde 3-phosphate dehydrogenase; however, NAD, 3-acetylpyridine-NAD, or thionicotinamide-NAD does not produce an acid-stable complex. Incubation of the glyceraldehyde 3-phosphate dehydrogenase with glyceraldehyde 3-phosphate, acetyl phosphate, iodoacetic acid, or iodosobenzoate inhibits the formation of the acid-stable complex with 3-pyridinealdehyde-NAD. Glyceraldehyde 3-phosphate or 3-pyridinealdehyde-NAD also prevents carboxymethylation of the active site cysteine-149 by[14-C]iodoacetic acid. These studies indicate that the aldehyde group of 3-pyridinealdehyde-NAD forms a thiohemiacetal linkage with cysteine-149 which is the substrate binding site for the dehydrogenase reaction. These findings may account for the fact that 3-pyridinealdehyde-NAD strongly inhibits the dehydrogenase and esterase activities of 3-pyridinealdehyde-NAD forms a thiohemiacetal linkage with cysteine-149 which is the substrate binding site for the dehydrogenase reaction. These findings may account for the fact that 3-pyridinealdehyde-NAD strongly inhibits the dehydrogenase and esterase activities of glyceraldehyde 3-phosphate dehydrogenase which require reduced cysteine-149. However, the analogue does not inhibit the acetyl phosphates activity of the enzyme for which the active site sulfhydryl residues must be oxidized.

The molar ratio of bound 3-pyridinealdehyde-NAD to the enzyme is 2.5 to 2.9. Lactate, malate, and alcohol dehydrogenases do not form acid-precipitable complexes with 3-pyridinealdehyde-NAD.
Incubation of the glyceraldehyde 3-phosphate dehydrogenase with glyceraldehyde j-phosphate, acetyl phosphate, iodoacetic acid, or iodosobenzoate inhibits the formation of the acid-stable complex with 3-pyridinealdehyde-NAD. Glyceraldehyde 3-phosphate or 3-pyridinealdehyde-NAD also prevents carboxymethylation of the active site cysteine-149 by [14C]iodoacetic acid. These studies indicate that the aldehyde group of 3-pyridinealdehyde-NAD forms a thiohemiacetal linkage with cysteine-149 which is the substrate binding site for the dehydrogenase reaction. These tidings may account for the fact that d-pyridinealdehyde-NAD strongly inhibits the dehydrogenase and esterase activities of glyceraldehyde 3-phosphate dehydrogenase which require reduced cysteine-149. However, the analogue does not inhibit the acetyl phosphatase activity of the enzyme for which the active site sulfhydryl residues must be oxidized.
Rabbit muscle glyceraldehyde S-phosphate dehydrogenase is known to bind the coenzyme NAD approximately 1000 times more tightly than do other dehydrogenases (1). The rabbit muscle enzyme crystallizes with 3 to 4 mol of NAD bound to each * This work was supported by Grants GM07884 and NS-10175 from the United States Public Health Service and grants from the National Science Foundation and the Muscular Dystrophy Association of America. mole of enzyme (2,3). This enzyme-coenzyme complex is not dissociated by dialysis (4) or by passing it through Sephadex columns (5). However, there are several reasons for believing the coenzyme is not bound covalently to the protein.
First, charcoal treatment of the enzyme complex can remove the coenzyme (4). In addition, [32P]NAD has been shown to exchange with bound NAD (6). Finally, when the conformation of the binding site is disturbed by acid precipitation, the enzyme-coenzyme complex dissociates.
In this paper, the nature of the NAD binding interactions is investigated by the use of NAD analogues.
Specific modifications of the P\'AD molecule give clues to the importance of particular atoms in NAD binding and the reactivity of the active site of this enzyme. In particular, the NAD analogue 3-pyridinealdehyde-NAD produces an acid-stable enzyme-coenzyme complex. The substrate, glyceraldehyde 3-phosphate, also forms an acidstable enzyme-substrate complex.
Both of these complexes prevent carboxymethylation of cysteine-149 in the active site of this enzyme, suggesting that each of these compounds forms a thiohemiacetal bond in the active site. The nature of the 3-pyridinealdehyde-NAD complex has been characterized, and the effects of this analogue on the dehydrogenase, esterase, and acetyl phosphatase activities of this enzyme have been investigated.

MATERIALS AND METHODS
Enzymes and Proteins-Glyceraldehyde 3-phosphate dehydrogenase from rabbit muscle was recrystallized at least three times in the presence of 1 mM EDTA by the method of Cori et al. (2). Enzyme-bound NAD was removed with activated charcoal (5) and the apoenzyme was dialyzed for at least 235 hours against 5 mM Tris-1 mM EDTA buffer, pH 6.8, in order to remove ammonium sulfate.
The apoenzyme gave a ratio of optical density readings at 280 and 260 nm which varied from 1.75 to 1.9. The enzyme concentration was determined from the extinction coefficient at 280 nm (3) and the molecular weight of the enzyme was taken at 140,000 (3).
Malate dehydrogenase was obtained from Boehringer Mannheim. Alcohol dehydrogenase was purchased from Worthington Biochemical Corp. Lactate dehydrogenase, aldolase, and papain were purchased from Sigma Chemical Co. Bovine albumin was obtained from Armour Laboratories.      (Table II).
In this study, four dehydrogenases, the glycolytic enzyme aldolase, the sulfhydryl protease papain, and serum albumin were selected. Kane of these proteins showed any 3-pyridinealdehyde-NAD binding after acid precipitation.

Effects of Xuljhydryl Inhibitors and Acetyl
Phosphate on S-

Pyridinealdehyde-NAD
Binding-Sodium tetrathionate has been shown to react specifically with cysteine residues in the active site of muscle glyceraldehyde 3-phosphate dehydrogenase (10, 11). Preincubation of the enzyme with 4 eq of sodium tetrathionate completely inhibited 3-pyridinealdehyde-NAD binding as shown in Table III.
The effects of two other sulfhydryl inhibitors, iodoacetic acid and iodosobenzoate, were investigated Dialyzed, NAD-free glyceraldehyde 3-phosphate dehydrogenase (0.03 pmol) was incubated in 0.1 M Tris buffer, pH 7.0, at 0" for 15 min after each addition as indicated below.
The total volume was 1.0 ml; 1.2 pmol of iodoacetic acid and iodosobenzoate and 0.12 rmol of 3-pyridinealdehyde-NAD were used. The bound 3-pyridinealdehyde-NAD was determined spectrophotometrically as described in Table  I.  (Table IV). Iodoacetic acid carboxymethylates only the reactive cysteine-149 in the active site of this dehydrogenase, whereas iodosobenzoate reacts first with the active site cysteine and subsequently oxidizes other -SH residues (11-13). As shown in Table IV, when iodoacetic acid or iodosobenzoate was added first, the inhibitor completely prevented the covalent 3pyridinealdehyde-NAD binding. However, when the 3-pyridinealdehyde-NAD was added first, iodoacetic acid displaced only 0.7 mol of the analogue, whereas iodosobenzoate removed 1.7 mol of the 2.5 mol of bound analogue.
In order to compare the analogue binding capacity of X-acetylated enzyme and N-acetylated enzyme, the pH dependence of acetyl phosphate inhibition of 3-pyridinealdehyde-NAD binding to the enzyme was investigated ( Table V). Inhibitions of 65% and 25% were observed at pH 4.5 and 7.0, respectively, whereas no inhibition was found at pH 8.5. Thus, it can be concluded that N-acetylation does not block analogue binding because Park et al. (5,14) have shown that at pH 4.5 an S-acetyl enzyme complex is formed with cysteine-149 and at pH 8.5 an N-acetyl enzyme complex is formed with lysine-185.
Inasmuch as acetyl phosphate forms a complex with only two catalytic sites at pH  Fig. 1. The apoenzyme and the enzyme preincubated with NAD both showed a final value of approximately 3.0 mol of [i4C]iodoacetic acid bound per mol of enzyme. The half-maximal alkylation time for the apoenzyme was 7 min. When the apoenzyme was preincubated with NAD, the well known (12, 15,16) enhancement of reactivity with iodoacetic acid With this technique, the apoenzyme bound 3.0 or 3.7 mol of NAD or 3-pyridinealdehyde-NAD per mol of enzyme, respectively (Table VI).
Preincubation of the apoenzyme with iodoacetic acid caused a 30y0 inhibition of the enzyme-coenzyme complex formation. Under the same conditions, iodosobenzoate produced approximately 50% inhibition of binding. If the enzyme-coenzyme complex was formed before the addition of the sulfhydryl inhibitors, the complex may be protected against dissociation.
This protection was stronger in the case of 3-pyridinealdehyde-NAD where iodoacetic acid or iodosobenzoate produced only a 3% or 20% dissociation of the preformed complex, respectively.
These data corroborate the results of the [Y$odoacetic acid binding studies in which NAD enhanced carboxymethylation, whereas 3-pyridinealdehyde-NAD strongly inhibited carboxymethylation. Effect of 3-Pyridinealdehyde-NAD on Kinetics of Dehydrogenase, Esterase, and Acety2 Phosphatase Reactions- Kaplan et al. (17,18) have shown that 3-pyridinealdehyde-NAD is a strong inhibitor of the dehydrogenase activity in the enzyme from muscle. With a ratio of 3-pyridinealdehyde-NAD to NAD of 1: 18, the dehydrogenase reaction is inhibited by more than 50%. inhibition was investigated by a kinetic study. A double reciprocal plot using a variable NAD concentration (Fig. 2) indicated that 3-pyridinealdehyde-NAD is a competitive inhibitor with respect to the coenzyme. A double reciprocal plot with a varying glyceraldehyde S-phosphate concentration was more complex (Fig. 3). As the 3-pyridinealdehyde-NAD concentration was increased, the K, for the substrate and the maximal velocity both increased.
The esterase reaction which splits p-nitrophenyl acetate to acetic acid and p-nitrophenol has been demonstrated by Park et al. (19). The effect of 3-pyridinealdehyde-NAD on this reaction was analogous to the effect of NAD, that is, stoichiometric inhibition of the rate of esterolysis by blocking acetylation of the enzyme.
The phosphatase activity of this enzyme is known to require both the presence of coenzyme and the oxidation of the cysteine-149 residue. Inasmuch as 3-pyridinealdehyde-NAD may bind to cysteine-149, one might expect that 3-pyridinealdehyde-NAD could produce phosphatase activity without requiring the addition of an oxidizing agent. Table VII shows that an oxidizing agent such as iodosobenzoate was still required to produce significant phosphatase activity.
Without iodosobenzoate, only negligible activity was elicited by the addition of either NAD or 3-pyridinealdehyde-NAD.
Although the activity with iodosobenzoate was reduced somewhat, 3-pyridinealdehyde-NAD did partially satisfy the coenzyme requirement for the phosphatase reaction.

DISCUSSION
An acid-stable complex was formed between rabbit muscle glyceraldehyde a-phosphate dehydrogenase and 3-pyridinealde- The reaction mixture and procedure were the same as those for Fig. 2 except that the NAD concentration was 0.60 mM and the subst,rate concentration was varied as shown on t,he abscissa.
The velocities of the reaction were measured on the control with no 3-pyridinealdehyde-NAD (O--O) and at 3.pyridinealdehyde-NAD concentrations cf 33 ELM (X--X) and 66 I.'M (A--A). The reaction mixture for the phosphatase assay contained the following: NAD-free glyceraldehyde 3-phosphate dehydrogenase, 0.005 rmol; iodosobenzoate, 1 pmol; NAD or S-pyridinealdehyde-NAD, 1 Kmol; acetyl phosphate, 6.7 pmol; and Tris buffer, pH 7.5, 100 pmol. Total volume was 2 ml. The NAD-free enzyme was incubated with coenzymes and iodosobenzoate as indicated in the table for 10 min at 0". The samples then were warmed to 25". The reaction was started by the addition of acetyl phosphate, and hydrolysis was allowed to proceed for 10 min. The disappearance of acetyl phosphate was quantitatively determined by the method of Lipmann and Tuttle (9). hyde-NAD, 3-pyridinealdehyde-deamino-NAD, and the substrate glyceraldehyde 3-phosphate. The coenzyme, NAD, and its analogues, thionicotinamide-NAD and 3-acetylpyridine-NAD, did not form acid-stable complexes with the dehydrogerlase. Each of the substances which formed an acid-stable enzyme complex contains an aldehyde group. This suggests that it is this aldehyde group which forms a covalent bond with the enzyme, possibly at the substrate binding site. The aldehyde groups on the NAD analogues are attached to carbon-3 on the nicotinamide ring. The hydrogens which are exchanged during the catalytic reaction are bound to carbon-4 of the nicotinamide ring. The known flexibility of the NAD molecule makes it possible for the NAD analogue to bond in the NAD binding site and still form this covalent linkage to the substrate binding site. NAD has been shown to be in the closed conformation with nicotinamide and adenine bases stacked when in solution (20). However, x-ray analysis has shown the coenzyme to be in the open conformation when bound to lactate dehydrogenase (21) and malate dehydrogenase (22).
The specificity of the 3-pyridinealdehyde-NAD covalent binding which was tested on a variety of proteins indicates that this complex is not an artifact due to trapping of the coenzyme or a general chemical reaction with an amino acid. This specificity is surprising for two reasons: first, because of the structural homology which is found between the different dehydrogenases; and second, because each of these dehydrogenases has a thiol residue which has been implicated as "essential" to the catalytic mechanism (23.-27). The structural homology was first shown between lactate dehydrogenase (28) and malate dehydrogenase (29). These two dehydrogenases have a very similar polypeptide backbone conformation throughout the entire molecule. Recently, horse liver alcohol dehydrogenase (30) and lobster glyceraldehyde 3-phosphate dehydrogenase (31) both have been shown to have a similar tertiary structure in the NAD binding area of the molecule.
Inasmuch as the coenzyme binding regions of these dehydrogenase proteins have similar three-dimensional structures and the coenzyme molecule itself has a flexible conformation which allows it to conform to the protein binding site, the specificity of the inhibitory effect of 3-pyridinealdehyde-NAD must result from a difference in spatial location or reactivity of the "essential" thiol residues in these dehydrogenase molecules.
It remains to be shown by peptide sequencing that 3-pyridinealdehyde-NAD does in fact bind to cysteine-149 in the rabbit muscle enzyme.
Rabbit muscle glyceraldehyde 3-phosphate dehydrogenase is inactivated irreversibly by alkylating reagents such as iodoacetic acid which react with the active site cysteine residue (2,13). It is reversibly inactivated by reagents which oxidize thiol residues, such as iodosobenzoate (32) and sodium tetrathionate (11,13). Competitive binding studies with each of these compounds indicate that the site of covalent binding of the aldehyde group is the same as the substrate binding site, cysteine-149. Sodium tetrathionate stoichiometrically inhibits 3-pyridinealdehyde-NAD binding, as would be predicted if they were competing for the same binding site (Table III). Preincubation with iodoacetic acid or iodosobenzoate inhibited the binding of j-pyridinealdehyde-NAD.
Iodosobenzoate will destroy the preformed acidstable complex, presumably by attacking the covalent bond, iodosobenzoate being a slightly stronger inhibitor under the conditions tested (Table IV).
Longer incubation times or higher incubation temperatures increase the displacement effect.
The studies with [Wliodoacetic acid showed that there is a slow time-dependent release of 3-pyridinealdehyde-KAD with binding of [14C]iodoacetic acid to cysteine-149 (Fig. 1). The 3-pyridinealdehyde-NAD displaced (approximately 10%) was less than that in Table IV due to variation in experimental conditions.
The inhibitory effect of 3-pyridinealdehyde-KAD on [14C]iodoacetic acid binding was essentially equivalent to that of the substrate, suggesting a similar bond.
Acetyl phosphate has been shown to exhibit a pH-dependent acetylation of the rabbit muscle enzyme (5,14). At pH 4.5 the acetyl group is predominately in a thioester linkage with cysteine-149. At pH 7.0 some acetyl groups bind to cysteine, whereas others bind to the e-amino group of a reactive lysine. At pH 8.5 the acetyl group is almost entirely in an N-acetyl linkage. Mathew et al. (14) have shown that the site of acetylation influences NAD binding.
Using a Sephadex G-25 column to separate free and bound NAD, they found 2.6 sites of NAD bound to the native enzyme or S-acetyl enzyme prepared with acetyl phosphate at pH 4.5; however, only 1.2 sites were bound to the Nacetyl enzyme formed with acetyl phosphate at pH 8.5. Thus, lysine-183 appears essential for maximal NAD binding.
Under the same conditions, 3-pyridinealdehyde-NAD has been shown to form an acid-stable complex with 2.6 sites of 3-pyridinealdehyde-NAD bound to the N-acetylated enzyme. This indicates that the thiohemiacetal bond formed in 3-pyridinealdehyde-NAD is so strong that lysine-183 is not necessary for binding.
The kinetic studies provide evidence about the effect of 3pyridinealdehyde-NAD on the dynamics of enzymatic catalysis. In the esterase reaction, 3-pyridinealdehyde-NAD and NAD both inhibited the initial step of acetylation of the enzyme by the substrate, p-nitrophenyl acetate. This suggests that both coenzymes bind at the same site and can sterically block the reaction of the large substrate with its phenyl ring.
The phosphatase reaction also implies that 3-pyridinealdehyde-NAD binds at the coenzyme binding site because bound coenzyme is known to be required for the phosphatase activity. The fact that iodosobenzoate is still required for activity supports the suggestion of l'arker and Allison (11) and Ehring and Colawick (34) that cysteine-149 must be oxidized to sulfenic acid for acetyl phosphatase activity.
The 30% reduction in activity with 3.pyridinealdehyde-NAD instead of NAD may be caused by competition between the aldehyde group of 3-pyridinealdehyde-NAD and iodosobenzoate to react with cysteine-149. If the apoenzyme is oxidized first, then the added NAD or 3-pyridinealdchyde-NAD is equally effective as the coenzgme in the phosphatase assay.
The analogue inhibition of the dehydrogenase reaction is very strong and somewhat complex because 3-pyridinealdehyde-NAD affects both the NAI) and the glyceraldchyde 3-phosphate binding sites. l'resumably the adenine end of a-pyridincaldehyde-NAD binds in the normal adenine binding site. The nicotinamide moiety positions itself so that the aldehyde group can react with cyst&e-149, forming a thiohemiacetal bond where the substrate normally forms such a bond.
Crystallographic studies' (35) as well as kinetic studies (36) indicate that it is the adenosine moiety of NAD which is important in coenzyme binding to the native conformation of dehydrogenase enzymes. The ultrafiltration experiments (Table VI) corroborate these studies. Both NAD and 3-pyridinealdehyde-SXI> bind to the native conformation of the enzyme. This should be expected because the adenosine portion of both molecules is the same and is bound to the same adenosine binding site.
However, under each condition tested in Table VI, 3-pyridinealdehyde-NAD showed a higher binding ratio than NAD. This implicates a difference in binding for the nicotinamide portions of these molecules, with aldehyde-nicotinamide having a higher binding affinity than nicotinamide.
The studies with the ultrafiltration method when combined with the acid precipitation data indicate that 3-pyridinealdehyde-NAD binds noncovalently to sites on the enzyme independent of the state of reduction of cysteine-149.
If this cysteine residue is reduced, the aldehyde group of 3-pyridinealdehyde-NAD also can form a thiohemiacetal linkage to the thiol residue at the substrate binding site. It is this covalent bond which produces the acidstable enzyme-coenzyme complex.