A Novel Diazonium-Sulfhydryl Reaction in the Inactivation of Yeast Alcohol Dehydrogenase by Diazotized 3-Aminopyridine Adenine Dinucleotide *

Diazotized 3-aminopyridine adenine dinucleotide has been found to modify four sulfhydryl groups per molecule of enzyme during the complete inactivation of yeast alcohol dehydrogenase. The reaction of sulfhydryl groups was indicated by titration studies with .5,5’-dithiobis(Z-nitrobenzoic acid) as well as isolation and quantitation of the cysteinyl derivative released by acid hydrolysis of the modified enzyme. The cysteinyl derivative was identified as S-(3-pyridyl)cysteine. Authentic S-(3-pyridyl)cysteine was synthesized and structurally characterized for these studies. Diazonium-sulfhydryl reactions were demonstrated for a number of diazonium derivatives with cysteine, homocysteine, glutathione, and mercaptoethanol at O-4” and neutral pH. Second order rate constants were determined in reactions of these sulfhydryl compounds with diazotized I-methyl-3-aminopyridinium chloride, diazotized 3-aminopyridine adenine dinucleotide, and diazotized j-aminopyridine adenine dinucleotide phosphate.

burg, Virginia 24061 SUMMARY Diazotized 3-aminopyridine adenine dinucleotide has been found to modify four sulfhydryl groups per molecule of enzyme during the complete inactivation of yeast alcohol dehydrogenase.
The reaction of sulfhydryl groups was indicated by titration studies with .5,5'-dithiobis(Z-nitrobenzoic acid) as well as isolation and quantitation of the cysteinyl derivative released by acid hydrolysis of the modified enzyme. The cysteinyl derivative was identified as S-(3-pyridyl)cysteine.
Authentic S-(3-pyridyl)cysteine was synthesized and structurally characterized for these studies.
Diazonium-sulfhydryl reactions were demonstrated for a number of diazonium derivatives with cysteine, homocysteine, glutathione, and mercaptoethanol at O-4" and neutral pH. Second order rate constants were determined in reactions of these sulfhydryl compounds with diazotized I-methyl-3-aminopyridinium chloride, diazotized 3-aminopyridine adenine dinucleotide, and diazotized j-aminopyridine adenine dinucleotide phosphate.
In recent studies (I), the chemical conversion of NAD to 3-aminopyridine adenine dinucleotide through the Hofmann hypobromite reaction was demonstrated to proceed mith a 68% yield.
The chemical, spectrophotometric, and flT.-rimetric properties of AADi were reported and, as an analog of NAD, this dinucleotide was shown to be a coenzyme-competitive inhibitor of several NAD-requiring enzymes (1). It was further observed that the 3aminopyridine moiety of AAD could be diazotized by reaction with nitrous acid and the resulting diazonium chloride could be azo-coupled with N-l-naphthylethylenediamine to form an azo dye.   (Fig. 3). From the slope of the line in Fig. 3 Thin layer chromatography of this minor component revealed two ninhydrin-positive spots, one of which corresponded to that of the major peak.
The fractions from the major peak were collected and lyophilized.
Thin layer chromatography of this product showed a single ultraviolet-quenching spot which was also ninhydrin positive, R F = 0.75 for the solvent system, 0.1 M acetic acid-95% ethanol (l:l, v/v), and RF = 0.65 for the solvent. system, butanol-acetic acid-water (5 :2 :3, v/v). Elemental analysis of carbon, hydrogen, and nitrogen supported the formula C&HloN202S. The melting point was 183-185" (uncorrected) with decomposition.
Analysis of the spectroscopic and chemical data indicated the compound to be the pyridy-1 thioether, S-(3.pyridyl)cysteine.
When the S-(3-pyridyl)cysteine was analyzed on the amino acid analyzer, with the use of a specially developed amino sugar program, a single peak was obtained at elution time 23.8 min (Fig. 5). The color factor, K F, was determined to be 0.4320.   ml of 60 PM yeast alcohol dehydrogenase in 0.10 M sodium phosphate buffer, pH 7.0, was added, and the mixture was incubated at O-4". Inactivation of the enzyme was monitored by assaying periodically for yeast alcohol dehydrogenase activity as detailed under "Methods." Inactivation of the enzyme was essentially complet,e by 15 min; however, the incubation was allowed to proceed for an additional 45 min. A control solution, lacking only AAD, showed no loss of enzyme activity during the same time interval.
Both sample and control solutions were dialyzed at 4' against five l-liter portions of 0.1 M sodium phosphate buffer, pH 7.0, over a 2-day period.
The ultraviolet difference spectrum of the modified versus the native yeast alcohol dehydrogenase was obtained. By assuming the molar extinction coefficient at 262 nm for the AAD residue on the modified enzyme to be the same as that for diazotized AAD, and correcting for 262-nm absorption due to yeast alcohol dehydrogenase, A peak corresponding to S-(3.pyridyl)cysteine (Fig. 5) was observed.
The concentration of X-(3.pyridyl)cysteine was seen to decrease linearly with increasing time of hydrolysis.
In order to det,ermine t,he concentrat,ion of the S-(3.pyridyl)cysteine residue present in the modified enzyme, extrapolation to zero time of hydrolysis was employed. Hgdrolysates of the unmodified enzyme did not contain any amino acid derivatives eluting after phenylalanine in the amino sugar program.
Synthetic S-(3-pyridyl)cysteine hydrolyzed for 24, 36, and 48 hours under conditions identical with those used for the acid hydrolysis of modified yeast alcohol dehydrogenase showed the same rate of destruction as that observed with S-(3.pyridyl). cysteine released from the modified enzyme.
This decay curve is very similar to that of methionine destruction in the acid hydrolysis of proteins. The number of moles of S-(3-pyridyl)cysteine released from inactivated yeast alcohol dehydrogenasc through acid hydrolysis (4 per tctrameric form of cnzymc) agreed well with the number of adenyl residues attached to the enzyme during the inactivation process. This is indicated by the comparison of spectral data and amino acid analysis data shown in Table II Positive chainlength effects in the inactivation of yeast alcohol dehydrogenase by N-alkylmaleimides indicated the importance of nonpolar interactions in reactions of sulfhydryl groups of this enzyme (14). The fact that NADH protected the enzyme against maleimide inactivation suggested that at least one of the functionally important sulfhydryl groups of the enzyme was located close to the hydrophobic region of the coenzymebinding site (19)(20)(21).
More recently, Twu and Wold (18) used butyl isocyanate to study the sensitive sulfhydryl groups of yeast alcohol dchydrogenase.
They reported that three sulfhydryl groups per molecule of enzyme were attacked during inactivation.
From peptide analysis, the modified sulfhydryl groups were shown to be different from those derivatized by iodoacetamide (22). Twu et al. (22) proposed that there are two distinct "essential" sulfhydryl groups per active site necessary for enzyme activity.
Although the reagents mentioned above can be selective for sulfhydryl groups, they are not necessarily site-directed reagents. However, since diazotized AAD is a structural analog of NAD, it can be preferentially bound at the active site of the enzyme. The parent compound AAD has also been found to be a coenzyme competitive inhibitor of yeast alcohol dehydrogenase (1). Thus, diazotized AAD is both active site directed and sulfhydryl group specific.
The fact that four sulfhydryl groups selectively react with diazotized AAD strengthens the argument that one of the functionally important sulfhydryl groups of yeast alcohol dehydrogenase is located nearby the pyridinium ring region of the coenzyme-binding site. Sloan and Mildvan (23), from magnetic resonance studies of the geometry of bound NAD and isobutyramide on spin-labeled yeast alcohol dehydrogenase, have also indicated that the spin label attached to cysteine is close to the dihydropyridine ring of bound NADH. Plapp et al. (24) in studies of the inactivation of yeast alcohol dehydrogenase by Ni-(w-bromoacetamidoethyl)nicotinamide also suggested the presence of a sulfhydryl group nearby the pyridinium ring region of the coenzyme-binding site. It will be interesting to identify the amino acid sequence of the peptide containing the diazotized AAD-modified sulfhydryl group and compare with results obtained by Harris (12) and Twu et al. (22). Such experiments are currently in progress. In view of the general lack of data concerning diazonium sulfhydryl reactions and the significance of this possibility for protein diazo coupling reactions, reactions with diazonium com-pounds were demonstrated with cystcine, homocystcine, and glutathione, indicating that sulfhydryl-containing amino acids and small peptides likewise react.
The essentially equivalent reactivity of mercaptoethanol confirms that the critical functional group for this reaction is the sulfhydryl group. The rates of reactions involving excess diazotized I-methyl-3.aminopyridinium chloride, diazotized XAD, and diazotized