Biochimica et Biophysica Acta, 787 (1984) 215-220 Elsevier
SULFHYDRYL ENZYME INACTIVATION BY NICOTINOYLACRYLATES B R U C E M. A N D E R S O N ~'*, C O N S T A N C E D. A N D E R S O N ~, G I O V A N N I D O N Z E L L I b and A L M A D A L POZZO b
u Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg, h Instituto Scientifico di Chimica e Biochimica, Giuliana Ronzoni, Milan (Italy)
VA 24061 (U.S.A.) and
(Received January 17th, 1984)
Key word~: Enzyme inactivation; Sulfhydryl group; Nicotinoylacrylate
Nicotinoylacrylic acid and the corresponding methyl, ethyl, propyl and benzyl esters were studied with respect to selective inactivation of dehydrogenases through covalent modification of essential sulfhydryl groups. An effective inactivation of yeast alcohol dehydrogenase, yeast glutathione reductase and yeast 6°phosphogluconate dehydrogenase was observed with methyl and ethyl nicotinoylacrylates, with nicotinoylacrylic acid and the larger propyl and benzyl esters being considerably less effective. Fluorescein mercuric acetate titration studies of inactivated yeast alcohol dehydrogenase and studies of the oxidized (disulfide) form of yeast glutathione reductase were consistent with inactivation processes involving sulfhydryl modification. Protection against nicotinoylacrylate inactivation was afforded by the binding of ligands to either the coenzyme or substrate binding sites. Inactivation of yeast alcohol dehydrogenase by ethyl nicotinoylacrylate exhibited an unexpected greater selectivity, resulting in the covalent modification of only one of the two reactive sulfhydryl groups at the catalytic site.
Introduction The presence of an a,fl-unsaturated carbonyl grouping has been recognized as important in the functioning of a variety of biologically active molecules . The biological functioning of such compounds has been related, in certain cases, to the reactivity of the a, fl-unsaturated carbonyl moiety towards nucleophilic groups of essential enzymes [2,3], and alkylation of enzyme functional groups [3,4] and small nucleophilic model compounds [5,6] has been reported. One class of a,flunsaturated carbonyl compounds, N-substituted maleimides, has received considerable attention with respect to enzyme inactivation through the selective covalent modification of enzyme functional groups. Although these reagents predominantly modify sulfhydryl groups of enzymes, reac* To whom correspondence should be addressed. 0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V.
tions with other enzyme functional groups can occur . A series of N-alkylmaleimides, varying in chainlength from methyl up to and including decyl, was synthesized  and used to investigate the relative nonpolarity of the environment of essential sulfhydryl groups of enzymes. In studies of dehydrogenases [8-11], sulfhydryl proteinases [12,13] and D-amino acid oxidase , rates of inactivation by N-alkylmaleimides were facilitated by nonpolar interactions with the enzyme sites involved. In contrast, the inactivation of three other dehydrogenases by these compounds was not facilitated by nonpolar interactions, which was related to the more polar nature of these enzyme sites [8,15]. In evaluating the effectiveness of these reagents in enzyme inactivation, it is important to recognize how the immediate environment of essential sulfhydryl groups can vary from one enzyme to another and can greatly influence rates of inactivation.
The importance of the a,fl-unsaturated carbonyl functionality prompted the synthesis and studies of numerous derivatives of fl-aroylacrylic acids [16-19], which were shown to have appreciable in vitro bacteriostatic and fungistatic activity. In a recent study , benzoylacrylic acid- and toluoylacrylic acid-derivatives were investigated for selective inactivation of three sulfhydryl enzymes known to exhibit different properties in sulfhydryl modification by N-alkylmaleimides. Yeast 6-phosphogluconate dehydrogenase, yeast alcohol dehydrogenase and yeast glutathione reductase show low, intermediate and high reactivity with N-alkylmaleimides, respectively. In the studies of benzoylacrylates , methyl esters were considerably more effective than the corresponding free acids in the inactivation of the alcohol dehydrogenase and glutathione reductase. This greater effectiveness of the methyl esters was attributed partly to an inherent higher reactivity toward simple sulfhydryl compounds such as cysteine, and partly to nonpolar interactions of the esters with the enzymes involved. The effectiveness of benzoyl- and toluoylacrylates in the inactivation of pyridine nucleotide-dependent enzymes suggested the possibility that more structurally related nicotinoyl derivatives may show an even greater selectivity in sulfhydryl modification and this prompted the present study of this class of compounds. In addition, nicotinoyl derivatives could serve as precursors for intracellular conversion to pyridine nucleotide analogs which would result in a more effective site-directed modification of dehydrogenases. Materials and Methods Materials
Fluorescein mercuric acetate, oxidized glutathione, NAD, NADP, NADPH, 6-phosphogluconate (trisodium salt), lithium lactate, glucose 6-phosphate and D-3-phosphoglyceric acid were purchased from Sigma. 3-Aminopyridine adenine dinucleotide phosphate was prepared by the method of Anderson et al. . Nicotinoylacrylic acid and the corresponding methyl, ethyl, propyl and benzyl esters were prepared according to Dal Pozzo et al. . Yeast glutathione reductase (NAD(P)H:oxid-
ized-glutathione oxidoreductase, EC 22.214.171.124) was obtained from Calbiochem-Behring. Yeast alcohol dehydrogenase (alcohol:NAD + oxidoreductase, EC 126.96.36.199), yeast 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP + 2oxidoreductase (decarboxylating), EC 188.8.131.52), Type V, rabbit muscle lactate dehydrogenase (Llactate:NAD + oxidoreductase, EC 184.108.40.206), rabbit muscle a-glycerophosphate dehydrogenase (sn-glycerol-3-phosphate:NAD + 2-oxidoreductase, EC 220.127.116.11), yeast glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP + 1oxidoreductase, EC 18.104.22.168), horse liver alcohol dehydrogenase (alcohol:NAD + oxidoreductase, EC 22.214.171.124) and chicken liver phosphoglycerate dehydrogenase (3-phosphoglycerate:NAD+ 2oxidoreductase, EC 126.96.36.199) were purchased from Sigma. Methods
Inactivation studies of glutathione reductase were performed by first preincubation 4/zg (3.410 -11 mol) enzyme at 25°C with a 500-fold excess of NADPH in a volume of 0.1 ml. To the reduced enzyme was added 0.9 ml 100 mM potassium phosphate buffer (pH 7.0) containing 5% ethanol and the desired concentration of nicotinoylacrylic acid derivative. After mixing, 50-~1 aliquots were assayed at timed intervals for remaining reductase activity. The activity was measured spectrophotometrically in 3-ml reaction mixtures containing 50 mM sodium phosphate buffer (pH 7.5)/1 mM EDTA/0.67 mM oxidized glutathione/0.1 mM NADPH. The decrease in absorbance at 340 nm was monitored at 25°C using a Beckman Acta MVI recording spectrophotometer. Inactivation studies of the remaining dehydrogenases did not require prior enzyme reduction and were performed at 25°C in 1 ml-reaction mixtures containing 100 mM potassium phosphate buffer (pH 7.0), 5% ethanol and the desired concentration of nicotinoylacrylic acid derivatives. Enzyme activities were measured spectrophotometrically at 340 nm using assay mixtures previously described [4,9,10,15]. Fluorescein mercuric acetate titration of sulfhydryl groups of native and inactivated yeast alcohol dehydrogenase was performed as described by Heitz and Anderson . Stock solutions of
fluorescein mercuric acetate were prepared in 0.1 M sodium pyrophosphate buffer (pH 8.0). Fluorescence titration of sulfhydryl groups was performed by recording relative fluorescence intensity using a Perkin-Elmer 650-40 spectrophotofluorometer. Solutions were excited at 495 nm and fluorescence emission recorded at 525 nm.
Ethyl nicotinoylacrylate at ~M concentrations was incubated with yeast alcohol dehydrogenase at 25°C. Samples were removed from the incubation mixture at timed intervals and assayed for enzyme activity. The enzyme became inactivated with time and the inactivation process followed pseudofirst-order kinetics as shown in Fig. 1. The pseudo-first-order rate constants calculated from these data were linearly related to ethyl nicotinoylacrylate concentration (Fig. 2). A second-order IOOL
40 I-Z W (J n~ W Q.
Fig. 1. Time-dependent inactivation of yeast alcohol dehydrogenase by ethyl nicotinoylacrylate. Reaction mixtures contained 6.4 ,ag enzyme in 100 mM potassium phosphate buffer (pH 7.0) and 5% ethanol in a total volume of 1 ml. The concentrations of ethyl nicotinoylacrylate used were 100 ,aM (line 1), 200 ,aM (line 2), 300 ,aM (line 3) and 400 ,aM (line 4).
200 300 [ETHYL NICOTINOYLACRYLATE](p.M}
Fig. 2. The effect of ethyl nicotinoylacrylate concentration on the pseudo-first-order rates of yeast alcohol dehydrogenase inactivation.
rate constant for inactivation of 270 M - 1 . min-1 was determined. Under the same conditions of incubation, reduced yeast glutathione reductase and yeast 6-phosphogluconate dehydrogenase were also observed to be inactivated by ethyl nicotinoylacrylate in pseudo-first-order reactions. The second-order rate constants for these inactivation processes are listed in Table I. Using the same conditions of incubation, inactivation of each of the above three enzymes was studied using nicotinoylacrylic acid and methyl nicotinoylacrylate (Table I). It can be noted that incubation of the oxidized form of yeast glutathione reductase (prior to coenzymatic reduction) with each of the nicotinoylacrylates had no effect on the catalytic activity of this enzyme. Propyl nicotinoylacrylate inactivation was observed but at considerably lower rates than those obtained with the methyl and ethyl esters. For example, the second-order rate constant for inactivation of glutathione reductase was estimated to be 100 M -1. min -1, 500-fold less than that determined for the methyl ester (Table I). Although solubility in 5% ethanol was not thought to be a factor, the inactivation was also studied in 20% DMSO and the same k z value was obtained. Similarly, a very slow rate of inactivation of glutathione reductase in 20% DMSO was observed with benzyl nicotinoylacrylate (Table I). Five other dehydrogenases were investigated for inactivation by ethyl nicotinoylacrylate. Rabbit muscle a-glycerophosphate dehydrogenase was inactivated by this derivative, yielding a second-order
218 TABLE I ENZYME INACTIVATION BY NICOTINOYLACRYLATES Enzyme
Yeast alcohol dehydrogenase Yeast glutathione reductase Yeast 6-phosphogluconate dehydrogenase
k 2 (M - i. min i ) Methyl ester
202 4990 1 000
270 4270 1045
340 b -
27 50 55
" Second-order rate constants determined at pH 7.0 and 25°C. b Second-order rate constant determined in 20% DMSO.
rate constant of 318 M - 1 . rain 1. Inactivation of chicken liver 3-phosphoglycerate dehydrogenase by ethyl nicotinoylacrylate exhibited saturation kinetics, and results were analyzed by the method of Kitz and Wilson . From double reciprocal plots, a maximum first-order rate constant of 0.077 min -1 and a K a value of 56/~M for ethyl nicotinoylacrylate were calculated. In contrast, rabbit muscle lactate dehydrogenase, horse liver alcohol dehydrogenase and yeast glucose-6-phosphate dehydrogenase were not inactivated by concentrations of ethyl nicotinoylacrylate up to 800 ~M. Four of the enzymes shown to be effectively inactivated by ethyl nicotinoylacrylate were observed to be protected from inactivation by selective ligand binding. The results of the protection experiments are shown in Table II. Yeast alcohol dehydrogenase was effectively protected by the binding of the coenzyme but not by the substrate. Similarly, yeast glutathione reductase was protected by the coenzyme analog, 3-aminopyridine adenine dinucleotide phosphate. The use of the coenzyme analog in this case was
necessary, since this analog is nonfunctional as a coenzyme and could not protect the reduced enzyme through a reoxidation process. 3-Aminopyridine adenine dinucleotide phosphate was previously demonstrated to be a coenzyme-competitive inhibitor of glutathione reductase . In contrast, 6-phosphogluconate dehydrogenase was protected by the binding of substrate and not by the coenzyme. In the case of 3-phosphoglycerate dehydrogenase, protection was observed with AMP, a substrate-competitive inhibitor. Fluorescence titration of the reactive sulfhydryl groups of native yeast alcohol dehydrogenase was performed using fluorescein mercuric acetate. As expected from many earlier studies, the results revealed the presence of eight reactive sulfhydryls per tool of enzyme or two sulfhydryls per subunit. The titration of yeast alcohol dehydrogenase, partially and totally inactivated by ethyl nicotinoylacrylate, is shown in Fig. 3. At 100% inactivation, four of the eight sulfhydryl groups, or one per subunit, had been modified. At 46% inactivation, 1.7 mol of sulfhydryl per mol of enzyme were
TABLE I1 PROTECTION AGAINST ETHYL NICOTINOYLACRYLATE INACTIVATION Enzyme
Ethyl nicotinoylacrylate (/~ M)
Yeast alcohol dehydrogenase Yeast 6-phosphogluconate dehydrogenase
Yeast glutathione reductase 3-Phosphoglycerate dehydrogenase a
NAD, 500 ~tM Phosphogluconate, I mM AADP, 100 #M AMP, 60 mM
Reactions run at pH 7.5
300' o g I O0
ACETATE (moles x I0 ~°)
Fig. 3. Fluorescence titration of yeast alcohol dehydrogenase with fluoresceinmercuric acetate. Reaction mixturescontained 0.1 M sodium pyrophosphate buffer (pH 8.0)/5% ethanol/33 ffM ethyl nicotinoylacrylate/fluoresceinmercuric acetate as indicated. Additions of enzymewere none (line 1). 4.18.10 al mol 100%-inactivatedenzyme (line 2) and 4.18.10-ll tool of 46%-inactivatedenzyme(line 3).
modified, or 0.42 sulfhydryl group per catalytic subunit. Discussion
The a,/~-unsaturated carbonyl grouping occurs in a number of biologically active compounds. The inhibition of the growth of microorganisms by this type of compound has been attributed to a facile covalent modification of nucleophilic functional groups of enzymes [1-6,18,24-27]. In a recent study of/Laroylacrylates , inactivation of yeast glutathione reductase and yeast alcohol dehydrogenase was demonstrated to occur through the selective modification of essential sulfhydryl groups. By comparing the rates of reactions of benzoyl- and toluoylacrylates with the sulfhydryl group of free cysteine, it is apparent that the protein environment of enzyme sulfhydryl groups can greatly influence the functioning of these compounds. The present studies were designed to gain further information about the functioning of such compounds with pyridine nucleotide-dependent enzymes. The nicotinoyl moiety was substituted for the previously studied benzoyl and toluoyl derivatives to provide a closer structural analogy to the pyridine nucleotide coenzymes. In general, rates of inactivation of dehydrogenases were enhanced by this substitution.
Yeast alcohol dehydrogenase, yeast glutathione reductase and yeast 6-phosphogluconate dehydrogenase were effectively inactivated by nicotinoylacrylates, with the methyl and ethyl esters showing considerably greater reactivity than the free acid (Table I). In comparison with the inactivation of these enzymes by methyl benzoylacrylate , methyl nicotinoylacrylate was more effective with the alcohol dehydrogenase, glutathione reductase and 6-phosphogluconate dehydrogenase by the factors 2.6, 7.6, and 91, respectively. This does not appear to be strictly related to the structural similarity of the nicotinoyl moiety of the acrylate reagent with that of the pyridine nucleotide coenzymes, since the greatest difference in inactivation rates occurred with 6-phosphogluconate dehydrogenase, where sulfhydryl modification occurs at the substrate binding site. The linear second-order plots observed (Fig. 2) indicated no prior binding of the nicotinoyl derivatives in these inactivation processes. In comparing rates of enzyme inactivation by nicotinoylacrylates, the higher reactivities observed with the methyl and ethyl esters are consistent with the results of previous enzyme studies  with benzoyl- and toluoylacrylates. The propyl and benzyl nicotinoylacrylates were considerably less effective. The ineffectiveness of these larger esters is thought to reflect negative steric interactions at the active sites of the enzymes studied. The inactivation of yeast alcohol dehydrogenase, yeast glutathione reductase and yeast 6phosphogluconate dehydrogenase by methyl benzoylacrylate was shown to be specifically related to the covalent modification of sulfhydryl groups at the active sites of these enzymes . The same mechanism of inactivation should apply in the reactions with nicotinoylacrylates. In the inactivation of yeast glutathione reductase by nicotinoylacrylates, modification of active-site sulfhydryls was suggested, since the oxidized (disulfide) form of the enzyme was not affected, and inactivation occurred only with the coenzymatically reduced (sulfhydryl) form of the enzyme. Sulfhydryl modification by ethyl nicotinoylacrylate was specifically investigated with yeast alcohol dehydrogenase. Titration of the native dehydrogenase with fluorescein mercuric acetate was performed to demonstrate the previously established [28-30]
presence of two reactive cysteine residues at the active site of the subunits of the enzyme. Fluorescence titration of enzyme completely inactivated by ethyl nicotinoylacrylate revealed the loss of one sulfhydryl group per subunit of enzyme. Correlation of the loss of enzyme activity with the modification of this sulfhydryl group was indicated by the observation that the modification of 0.42 sulfhydryl/subunit occurred at 46% inactivation. In general, methyl and ethyl nicotinoylacrylates were more effective than methyl benzoylacrylate in the inactivation of the enzymes studied. This is supported both by the higher second-order rate constants observed and by the slightly higher concentrations of ligands required for enzyme protection (Table II). Data are not available to compare the inherent reactivities of nicotinoylacrylates to those of benzoyl- or toluoylacrylates with respect to sulfhydryl addition reactions. However, in recent studies of the addition of piperidine to these different acrylates (unpublished results), the addition reactions with nicotinoylacrylates were 2-3fold faster than reactions with the corresponding aroylacrylates. If one assumes a similar higher reactivity of nicotinoylacrylates with other nucleophiles such as sulfhydryl groups, then, on the basis of inherent reactivity, one would expect enzyme inactivation by methyl nicotinoylacrylate to proceed at 2-3-times the rate of inactivation by methyl benzoylacrylate. The covalent modification of sulfhydryl groups in the nicotinoylacrylate inactivation of dehydrogenases is suggested by the above results; however, hydrolysis and product studies of inactivated enzymes would be required to rule out modification of other functional groups.
Acknowledgements These studies were supported by Research Grant PCM 82-06712 from the National Science Foundation and N A T O Research G r a n t No. RG036.80/D1.
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