Mechanism for the inhibition of aldehyde dehydrogenase by nitric oxide

Mechanism for the inhibition of aldehyde dehydrogenase by nitric oxide

Alcohol, Vol. 14, No. 2, pp. 181-189, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0741-8329/967 $17.00 + .00 ...

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Alcohol, Vol. 14, No. 2, pp. 181-189, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0741-8329/967 $17.00 + .00

PII S0741-8329(96)00142-5


Mechanism for the Inhibition of Aldehyde Dehydrogenase by Nitric Oxide EUGENE

G. D E M A S T E R , *




*Medical Research Laboratories, Department of Veteran Affairs Medical Center, Minneapolis, MN 55417 ?Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455 R e c e i v e d 8 M a y 1996; A c c e p t e d 25 J u l y 1996 DEMASTER, E. G., B. REDFERN, B. J. QUAST, T. DAHLSEID AND H. T. NAGASAWA. Mechanism for the inhibition of aldehyde dehydrogenase by nitricoxide. ALCOHOL 14(2) 181-189, 1997.-- The inhibition of SaccharomycescerevMae aldehyde dehydrogenase (A1DH) by gaseous nitric oxide (NO) in solution and by NO generated from diethylamine nonoate was time and concentration dependent. The presence of oxygen significantly reduced the extent of inhibition by NO, indicating that NO itself rather than an oxidation product of NO such as N203 is the inhibitory species under physiological conditions. A cysteine residue at the active site of the enzyme was implicated in this inhibition based on the following observations: a) NAD + and NADP +, but not reduced cofactors, significantly enhanced inhibition of A1DH by NO; b) the aldehyde substrate, benzaldehyde, blocked inhibition; and c) inhibition was accompanied by loss of free sulfhydryl groups on the enzyme. Activity of the NO-inactivated enzyme was readily restored by treatment with dithiothreitol (DTT), but not with GSH. This difference was attributed, in part, to a redox process leading to the formation of a cyclic DTT disulfide. Based on the chemistry deduced from model systems, the reaction of NO with A1DH sulfhydryls was shown to produce intramolecular disulfides and N20. These disulfides were shown to be intrasubunit disulfides by nonreducing SDS-PAGE analysis of the NOinhibited enzyme. Following complete inhibition of A1DH by NO, four of the eight titratable (Ellman's reagent) sulflaydryl groups of AIDH were found to be oxidized to disulfides. These results suggest that a) the sulfhydryl group of active site Cys302 and a proximal cysteine are oxidized to form an intrasubunit disulfide by NO; b) only two of the four subunits of AIDH are catalytically active: and c) NO preferentially oxidizes sulfhydryl groups of the catalytically active subunits. A detailed mechanism for the inhibition of AIDH by NO is presented, o 1997 Elsevier Science Inc. Aldehyde dehydrogenase

Nitric oxide

Inhibition of aldehyde dehydrogenase

I N C R E A S E D hepatic acetaldehyde ( A c H ) levels following chronic ethanol ingestion are believed to be etiological in alcoholic liver disease, because A c H is known to form adducts with liver proteins (31,42), enhances hepatic lipid peroxidation (41), and p r o m o t e s collagen synthesis-and fibrosis (23,32). H o w e v e r , early events leading to decreased hepatic aldehyde dehydrogenase (AIDH) activity (33,34,39) and increased hepatic A c H levels, evidenced by elevated blood A c H in alcoholics (35), are not understood. W e hypothesize that enhanced nitric oxide ( N O ) production (30) due to an increase in hepatocyte nitric oxide synthase (NOS2) with chronic alcohol use causes inhibition of hepatic A 1 D H in vivo, thereby elevating A c H . Inhibition of A I D H in vivo by N O is also suggested by reduced erythrocyte A 1 D H activity in patients treated with iso-

sorbide dinitrate and nitroglycerin (48) and by reduced hepatic A1DH activity following exposure to certain aldoximes (10,21). Organic nitrates and aldoximes are known metabolic precursors of N O (15,20). In related studies, induction of hepatocyte N O S 2 by Corynebacterium parvum caused a decrease in hepatic glyceraldehyde-3-phosphate dehydrogenase ( G A P D H ) activity in rats (27). In vitro studies indicate that N O inhibits G A P D H and p r o m o t e s the N A D + - d e p e n d e n t A D P ribosylation of Cys-149 (6), the active site sulfhydryl group. In the A D P ribosylation of G A P D H , an oxidation product of NO, presumably N~O3, reacts with the cysteine thiol to form an S-nitrosothiol, which is then acted on by enzyme-bound N A D + to yield the A D P ribosylated protein. Saccharomyces cerevisiae (yeast)

Requests for reprints should be addressed to Eugene G. DeMaster, Ph.D., Medical Research Laboratories (151), VA Medical Center, One Veterans Drive, Minneapolis, MN 55417. Tel: (612) 725-2000, ext. 2854 or 2828; Fax: (612) 725-2093. ~Based in part on the presentation, "Inhibition of yeast aldehyde dehydrogenase by nitric oxide," at the FASEB meeting in Anaheim, CA, April 26, 1994. 181



AIDH is also inhibited by NO (26). Unlike GAPDH, however, this yeast A1DH is modified by ADP-ribose via an NOindependent mechanism, and not by NAD + (26). Therefore, the inhibition of yeast AlDH by NO and related substances can be studied without interference from a secondary inhibition by NAD+-dependent A D P ribosylation. The rationale for using yeast A1DH as a model for the mammalian mitochondrial A1DH isozyme is based on the similarities of their kinetic properties and reaction mechanisms (14), the conservation of key amino acid residues at the active site believed essential for activity (40), as well as the lack of a secondary inhibition (i.e., NAD ~-dependent A D P ribosylation). Furthermore, substances that inhibit yeast A1DH also inhibit the mammalian low K m hepatic AIDH, and vice versa (10-12,52). In this report, the inhibitions of A1DH by gaseous NO in solution and by NO generated from diethylamine nonoate (DEA/NO) are shown to be similar in their response to a) aerobic and anaerobic conditions, b) oxidized and reduced pyridine nucleotides, c) thiol protective agents, and d) thiols that reverse this inhibition. Evidence is presented that the NO-inhibited form of the enzyme is a disulfide rather than an S-nitrosothiol derivative of the active site cysteine.


Materials Dithiothreitol (DTT), GSH, NADH, mercaptoethanol, and activated charcoal were purchased from Sigma Chemical (St. Louis, MO). Stock solutions of NO (approximately 2.0 raM) were prepared in deionized water from NO gas (Matheson Gas Products, Chicago, IL) as previously described (2). DEA/NO and N-nitrosohydroxylamine-sulfonate (SULFI/NO) were generously provided by Dr. Larry Keefer, NCI, NIH. Angeli's salt and isobutyraldehyde oxime were synthesized as described by Hunt et al. (18) and Vogel (50), respectively. Stock solutions of DEA/NO, SULF1/NO, and Angeli's salt were prepared in deoxygenated 10 mM KOH. Caution: NO gas is highly toxic, and therefore, the preparation of the 2.0 mM stock solution of gaseous NO must be prepared in a fumehood! Yeast AIDH (EC was obtained from Boehringer Mannheim Corp. (Indianapolis, IN) and dialyzed against 20 mM potassium phosphate buffer (pH 7.4) containing 0.2 M KC1 at 4°C in a nitrogen atmosphere for 4 h or overnight before use. Cofactor-free enzyme was prepared using neutralized activated charcoal as described by Taylor et al. (47). AIDH concentration is based on protein content, which was determined using bicinchoninic acid with human serum albumin as the standard (43) and a molecular weight of 224,000 (40).

Inhibition Studies The effect of NO-based inhibitors on the activity of yeast AIDH was assessed using a two-step assay system as previously described (28). The primary reactions were carried out in 0.75-ml sealed reaction vessels under anaerobic conditions, unless noted otherwise. The thiols, A1DH, gaseous NO solution, and D E A / N O solutions were added through the septa of the reaction vessel by syringe. For anaerobic conditions, all solutions were deoxygenated with argon before use. The standard primary reaction mixtures contained 100 mM potassium phosphate (pH 7.4) and 1.0 mM NAD +, where indicated, were preincubated for 5 rain at 37°C (deoxygenated using a stream of argon during the last 3 rain of the preincubation period) followed by the addition of 0.08 U yeast A1DH and 0-10

ixl of inhibitor solution for a total volume of 0.1 ml in sealed reaction vials (Pierce, Rockford, IL). After incubation periods of 3, 6 or 10 rain, a 20-txl aliquot of the primary mix was removed and added to a cuvette containing 0.5 mM NAD +, 1.0 mM EDTA, 30% glycerol, and 100 mM potassium phosphate (pH 8.0) in a final volume of 1.0 ml. This secondary reaction was initiated by the addition of benzaldehyde (0.6 ixmol) and carried out at 25°C. The activity of yeast AIDH was determined spectrophotometrically by following the increase in NADH (340 nm) over time.

Analysis of End Products Hydroxylamine derived from nitroxyl, a reaction intermediate, was analyzed as isobutyraldehyde oxime using isobutyraldehyde as the trapping agent. This oxime derivative was quantified by headspace GC using flame ionization detection. Sodium chloride (0.8 g) was added to the GC vials to enhance the partitioning of the oxime into the headspace. The samples were heated at 70°C for 30 min in the aluminum heating block of the autosampler (Perkin Elmer Corporation, model HS-100) before injection onto the column. The GC conditions were as follows: column, 2 mm (inner diameter) × 2 m glass packed with Tenax-GC, 80-100 mesh (Supelco, Inc., Bellefonte, PA); column temperature, 160°C; carrier gas, N:; flow rate, 40 ml/min. Under these conditions, the retention times for isobutyraldehyde and its oxime were 0.98 and 2.43 rain, respectively. Oxime values were based on peak areas and were calculated by use of an external standard. The lower limit of measurement was 1.0 gM. N,O was measured by GC with thermal conductivity deteclion as previously described (29). NzO standards were prepared by addition of known amounts of N20 (935 ppm prepared in He was purchased from Matheson Gas Products) using a gastight syringe to 22-ml glass septum vials containing 1.0 ml of 0.1 M potassium phosphate buffer, pH 7.5. These standards were heated at 37°C for 15 min, and 0.6 ml of headspace was analyzed. The retention time for N2O was 2.7 rain and quantitation of N,O was based on peak area.

NO Analysis NO in the headspace of reaction mixtures was measured by chemiluminescence using a Sievers NO Analyzer (Boulder, CO) as previously described (21). NO (10 ppm) in He (Matheson Gas Products) was used as an external standard.

Electrophoresis Nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the method of Laemmli (22) with a 4% stacking gel and 12% separating gel, but omitting mercaptoethanol from the sample buffer. The gels were stained with Coomassie Blue.

Fluorescence Measurements AIDH was pretreated with and without DEA/NO as described in Table 4 followed by extensive dialysis to remove NAD +. The fluorescence measurements were carried out using a SLM Aminco SPF-500C spectrofluorometer at 22°C and a 1.0-cm square cuvette. The fluorescence spectra of 0.1 mM N A D H excited at 375 nm in 0.1 M potassium phosphate, pH 7.0, with and without active or NO-inhibited AIDH (2.0 nmol) in final volume of 2.0 ml was recorded from 400 to 550 nm.


Data Presentation Experimental values are given as means + SEM of triplicate samples, unless indicated otherwise. Statistical comparisons were made using the Student's t-test and a value of p < 0.05 was accepted as significant. RESULTS

Inhibition of Yeast AIDH by NO Time courses for the inhibition of A1DH by gaseous NO in solution and by NO derived from D E A / N O are shown in Fig. 1. With aqueous solutions of NO, the time course of inhibition showed a large initial loss of A1DH activity followed by a slower rate of inhibition (Fig. 1, upper panel). This biphasic time course is attributed to the rapid partitioning of the added NO into the headspace of the reaction vessel (2). The extent of AIDH inhibition by NO was highly dependent on the presence of N A D +. In contrast, D E A / N O , which has a half-life of 2.1 min under these conditions (25), slowly releases NO into the media via a first-order process. Thus, the time courses for the inhibition of A I D H by D E A / N O (Fig. 1, lower panel) lack the biphasic properties observed with gaseous NO. In most of the subsequent experiments with gaseous NO and D E A fNO, the reaction times of 3.0 and 6.0 min were used, respectively. As illustrated in the time course study (Fig. 1), N A D + significantly enhanced the inhibition of A1DH by NO. Using a range of inhibitor concentrations, the respective ICs0 values for the inhibition of A I D H by gaseous NO and D E A / N O were

found to be 150 and 135 I~M in the absence of N A D ÷ and 39 and 42 IxM in the presence of 1.0 mM N A D +. The degree of enhancement of the NO inhibition of A1DH by N A D + was dependent on the N A D + concentration (Fig. 2). The calculated dissociation constant for N A D + in this process was 20.0 ~M. N A D P +, an alternative cofactor for yeast A1DH (44), similarly enhanced the NO inhibition of A1DH; however, the reduced forms of these cofactors showed no enhancement effect (data not shown). Although commercial preparations of yeast A1DH are not known to contain N A D + or N A D P +, one preparation was pretreated with charcoal to insure a cofactor-free form of the enzyme. A q u e o u s solutions of gaseous N O inhibited this cofactor-free enzyme to the same degree as untreated AIDH preparations without added cofactor. Inhibition of A I D H by gaseous NO and D E A / N O under aerobic vs. anaerobic conditions. When D E A / N O was added to reaction mixtures containing A I D H under aerobic conditions and compared with similar reaction mixtures under anaerobic conditions, the concentration-inhibition curve for the effect of D E A / N O on A I D H activity was shifted to the right (Fig. 3). However, at lower concentrations, a crossover was observed, suggesting the presence of another inhibitory species under these conditions (i.e., a species more inhibitory than NO but short-lived). The results obtained with gaseous NO (data not shown) were nearly identical to those shown for D E A / N O .

Blockade of NO Inhibition of AIDH by Benzaldehyde Because the reaction catalyzed by yeast A1DH proceeds by an ordered mechanism (14) (i.e., N A D + binds to the enzyme before the aldehyde substrate), benzaldehyde would be expected to block enzyme inhibition by NO in the presence, but not absence, of N A D +. Indeed, benzaldehyde (5.0 mM) effectively blocked the inhibition of A I D H by gaseous NO (200 txM) in the presence of N A D + (Fig. 4), whereas in the absence of N A D +, benzaldehyde only partially blocked inhibition by NO. Likewise, this same concentration of benzaldehyde almost completely blocked the inhibition of A I D H by D E A / N O (150 txM) in the presence of N A D +, but did not block the D E A / N O inhibition of A1DH in the absence of N A D + (data not

1001 80 60 --~ 4O

= ~ 20 O





L, ~a

~u 8O





0.06 ~0.04





•~ 0.00





"" 20

0 0













Time (min) FIG. 1. Time courses for the inhibition of A1DH by 200 ixM gaseous NO (upper panel) and DEA/NO (lower panel) with (closed symbols) and without (open symbols) NAD +. The concentrations of DEA/NO were 75 (O, Q) and 200 p,M ( A A). Each time course represents multiple sampling from a single reaction mixture. Other experimental details are given in the Method section.

NAD + (mM) FIG. 2. Effect of NAD + concentration on the inhibition of AIDH by 0.0 (O) and 200 p~M (O) gaseous NO. Experimental details were as described in Fig. 1. The double reciprocal plot of the NAD + concentration vs. the remaining A1DH activity is given in the inset. The Ks for NAD + was calculated to be 20 ~M.



Reactivation q[ NO-inhibited A IDtt by Thiols


T h e reactivation of i n h i b i t e d A I D H was assessed using G S H a n d D T T on the N O - t r e a t e d enzyme. G S H was minireally effective in restoring activity to the i n h i b i t e d enzyme, w h e r e a s 69 to 98 p e r c e n t of the lost activity was restored by D T T t r e a t m e n t u n d e r the conditions used in T a b l e 1. However, w h e n the G S H concentration was increased to 20 raM, G S H restored 12 _+ 1% of activity to N O - i n h i b i t e d A I D H after 3.0 rain and 75 -+ 2 % after 30 rain of t r e a t m e n t . Similar results were o b t a i n e d with equimolar amounts of mercaptoethanol. T h e differential activity of D T T vs. G S H a n d / o r m e r c a p t o e t h a n o l in restoring e n z y m e activity is a t t r i b u t e d , in part, to t h e i r respective sulfhydryl c o n t e n t and, in part, to their redox potentials (also see Discussion).

80 60


40 20

0 0

100 DEA/NO

200 (~M)

Reactions o# D TT With NO, Thionitrite, and Nitroxyl (HNO)

FIG. 3. Relation of DEA/NO concentration to inhibition of AIDH activity under aerobic (©) and anaerobic (@) conditions. NAD ~ concentration was 1.0 raM. Other experimental details were as described in Fig. 1. ICs0 values for DEA/NO under aerobic and anaerobic conditions were 139 and 51 /xM, respectively.

shown). T h e differential b l o c k a d e by b e n z a l d e h y d e of A I D H inhibition by D E A / N O derived N O a n d by gaseous N O in the a b s e n c e of N A D + can b e a t t r i b u t e d to the differences in N O c o n c e n t r a t i o n in solution following the a d d i t i o n of D E A / N O a n d gaseous N O .

Blockade of the NO Inhibition of AIDH by Thiols G S H and D T F were evaluated for their ability to competitively sequester N O a n d thus block the i n h i b i t i o n of A I D H by gaseous N O a n d D E A / N O . D T T almost completely b l o c k e d inhibition of A 1 D H by NO. In contrast, the G S H b l o c k a d e of A I D H was variable with g r e a t e r p r o t e c t i o n o b s e r v e d in the a b s e n c e than in the p r e s e n c e of N A D + ( T a b l e 1). E v e n w h e n the thiol c o n c e n t r a t i o n of G S H was i n c r e a s e d to 20 m M (i.e., e q u i m o l a r in sulfhydryl c o n t e n t to 10 m M D T F ) , D T F was still substantially m o r e p r o t e c t i v e t h a n G S H ( d a t a not shown).

T o gain a b e t t e r u n d e r s t a n d i n g of the reactions of N O with vicinal a n d / o r proximal thiols, D E A / N O was i n c u b a t e d with D T T and the reaction mixtures were analyzed for hydroxyla m i n e ( T a b l e 2) and N 2 0 (Table 3), the possible end products of N O r e d u c t i o n by DTT. T h e h y d r o x y l a m i n e p r o d u c e d was t r a p p e d with i s o b u t y r a l d e h y d e [eq. (1)] and was m e a s u r e d as (CH3)eCH-CH = O + H2NOH-->(CH3)eCH-CH = NOH + H~O

( 1)

the oxime of this a l d e h y d e by h e a d s p a c e G C analysis. Because the N O inhibited form of A I D H was considered to be possibly a thionitrite, the r e a c t i o n of S - n i t r o s o g l u t a t h i o n e ( G S - N O ) with D T T was also investigated. M o r e o v e r , as nitroxyl was a p o s t u l a t e d p r o d u c t of this reaction, the effect of D T T on nitroxyl g e n e r a t e d from A n g e l i ' s salt (Na2N20.;) was also studied. T h e immediate decomposition products of Angeli's salt are nitroxyl a n d nitrite. Nitroxyl s u b s e q u e n t l y dimerizes to h y p o n i t r o u s acid ( H O N = N O H ) a n d the latter rapidly deh y d r a t e s to yield N ; O (4,17). S U L F I / N O , which d e c o m p o s e s directly to h y p o n i t r o u s acid w i t h o u t going t h r o u g h a nitroxyl i n t e r m e d i a t e (38,45) via a first-order process with a half-life of 6.8 rain u n d e r physiological conditions (25), was used as control.





Prevention of Inhibition (%)*

60 Thiol (10 mM)

NAD+ (1.0mM)

Gaseous NO (20 nmol)

DEA/NO (15 nmol)

60 + 8 25 ± 5 91) + 5 99 + 0.3

25+ 8 9+ 1 100 +_ 2 87 _+ I

Reversal of lnhibitkm t % )" Gaseous NO (20 nmol)

DEA/NO (15 nmol)


~ ,--


20 I







1 2 3 4 Benzaldehyde (raM)




FIG. 4. Benzaldchyde effectively protects AIDH from inhibition by gaseous NO (200 IxM) in the presence (O), but not absence (O), of NAD +. Benzaldehyde was added to the primary incubation mix before the addition of NO. Note that in the absence of NAD + only 50% inhibition of the enzyme was observed.

+ +

5 +4 8 ± 0.4 69 + 16 87 + 2

<5 12 + 1 98 ± 5 86 ± 2

The experimental conditions were as in Fig. 1, except the thiols were added to the primary mixture 15 s before AIDH for prevention of inhibition. For reversal of inhibition, the Ihiols were added to the primary mix after 3 rain of incubation with gaseous NO and alter 6 rain of incubation with DEA/NO. Aliquots of the primary mix for AIDH activity measurements were taken 3 rain after thiol addition. *Prevention or reversal of inhibition (%) = (% inhibition of A1DH without thiol minus % inhibition with thiol) divided by % inhibition of AtDH without thiol.



TABLE 2 REDUCTION OF NO AND HNO FROM GENERATORS, AND OF GS-NO BY DTT TO HYDROXYLAMINE Hydroxylamine Produced (nmol)* Donor Substance (50 n m o l ) 1. 2. 3. 4.


Conditions Aerobic Anaerobic Aerobic Aerobic Aerobic

pH 7.5

pH 9.0

7.4 + 0.6 <0.5 22.9 ± 0.3 49.2 _+ 1.3 <0.5

5.9 + 1.1 1.6 ± 0.2 39.3 ± 2.3 44.8 ± 2.4 <0.5

Reaction mixtures containing 40 mM potassium phosphate buffer, pH 7.5, or 40 mM sodium pyrophosphate, pH 9.0, with 100 mM potassium chloride were preincubated for 5 min at 37°C, which was followed by the addition of 5 ixmol DTT, 0.25 ixmol isobutraldehyde, and 50 nmol donor in a final volume of 0.5 ml in 22-ml glass septum vials. Reactions with GS-NO and DEA/NO were incubated for 15 min, SULFI/NO for 30 mira and Angeli's salt for 60 min, all at 37°C. After incubation, the samples were placed on ice, and the isobutyraldoxime product was measured by headspace GC as described in the Method section. * Values are based on n = 4. ?One tool of DEA/NO decomposes to 1 tool of diethylamine and 2 tool of NO in a first-order reaction with a half-life of 2.1 min at pH 7.4 and 37°C (25).


v e r t e d to N 2 0 (Table 3) with the r e m a i n d e r of the N O f o u n d in the h e a d s p a c e of the reaction vessel after 1 h of incubation (data not shown). DTI" r e d u c e d a b o u t 50% and 80% of the a d d e d G S - N O to hydroxylamine at p H 7.5 and 9.0, respectively (Table 2), whereas no N 2 0 was p r o d u c e d f r o m G S - N O in either the p r e s e n c e or a b s e n c e of D T F (Table 3). T h e s e results d e m o n s t r a t e that the nitroxyl intermediate generated by the DTI" reduction of GS-NO was r e d u c e d f u r t h e r to h y d r o x y l a m i n e by the excess DTT. This was c o r r o b o r a t e d by the o b s e r v a t i o n that the nitroxyl f o r m e d f r o m A n g e l i ' s salt was almost quantitatively r e d u c e d by D T F to h y d r o x y l a m i n e (Table 2), w h e r e a s without D T T , nitroxyl d i m e r i z e d and p r o d u c e d N 2 0 (Table 3). Because S U L F I / N O d e c o m p o s e s directly to h y p o n i t r o u s acid without the i n t e r m e d i a c y of nitroxyl, the f o r m a t i o n of its m a j o r e n d product, N20, was only minimally affected by the p r e s e n c e of D T T (Table 3).

Characterization of NO-Inhibited AID H

N o h y d r o x y l a m i n e was p r o d u c e d f r o m the reaction of D E A / N O - d e r i v e d N O with D T T u n d e r a n a e r o b i c conditions at p H 7.5 (Table 2). H o w e v e r , u n d e r aerobic conditions, 7.4% of the N O p r o d u c e d was c o n v e r t e d to the oxime. T h e hydroxylamine p r o d u c e d u n d e r aerobic conditions was a t t r i b u t e d to the f o r m a t i o n of N203, which can nitrosylate D T T to f o r m an S-nitroso thiol (vide infra). U n d e r a n a e r o b i c conditions ( p H 7.5), a b o u t 50% of the a d d e d D E A / N O - d e r i v e d N O was con-

Y e a s t A I D H is k n o w n to have eight readily titratable sulfhydryl groups, two p e r subunit (5), which c o m p a r e s with 7.5 mol sulfhydryl groups per mol e n z y m e found in our experim e n t (Table 4). W h e n A 1 D H was e x p o s e d to 200 IxM D E A / NO, a c o n c e n t r a t i o n that c o m p l e t e l y inhibits A1DH (Fig. 1), a b o u t half of these sulfhydryl groups w e r e oxidized by N O (i.e., 3.6 mol sulfhydryl oxidized per mol enzyme). T h e e n d p r o d u c t s of N O oxidation of a p r o t e i n thiol are either a sulfenic acid and N20 or a disulfide and N20, depending on steric factors (13). The s t o i c h i o m e t r y for t h e s e reactions in t e r m s of sulfhydryl oxidized vs. N 2 0 p r o d u c e d are 1:1 and 2:1, respectively (see Discussion). Following the inhibition of A1DH by N O , the ratio of A1DH sulfhydryls oxidized to N20 p r o d u c e d was nearly 2 (Table 4), indicating that an i n t r a m o lecular disulfide was f o r m e d . A l t h o u g h each subunit of A1DH contains two free cysteine sulfhydryl groups j u x t a p o s i t i o n e d to one a n o t h e r (19), an int r a m o l e c u l a r disulfide b o n d could f o r m either b e t w e e n two cysteine residues of the same subunit or b e t w e e n two cysteine



1. 2. 3. 4.

Donor Substance (100 nmol)

DTT (10 raM)

Nitrous Oxide Produced (nmol produced at pH 7.5)

DEA/NO DEA/NO GS-NO GS-NO Angeli's salt Angeli's salt SULF1/NO SULFI/NO

+ +

<1.0 53.5 -+ 3.2 <1.0
+ +

Reaction mixtures containing 50 mM potassium phosphate buffer, pH 7.5, in 22-ml glass septum vials were deoxgenated with a stream of argon. The reactions were initiated by the addition of 10 ixmol of DTT (deoxgenated) where indicated followed by 100 nmol of donor substance in a final volume of 1.0 ml. Reactions with GS-NO were incubated for 30 rain, whereas the others were incubated for 1 h, all at 37°C. After incubation, a 0.6-ml aliquot of the sample headspace was immediately analyzed for N20 content by GC as described in the Method section.

DEA/NO Added (nmol)

AIDH SH* (nmol)

NzO Produced (nmol)

SH Oxidized N20 Produced

0.0 200

17.7 -+ 0.5 9.3 ± 0.2

<0.5 4.4 _+ 0.4?


The incubation and experimental conditions were as described in Fig. 1 (lower panel) except the reaction mixtures were scaled up 10 fold and contained 2.37 nmol A1DH. The volume of the reaction vessels was 6.1 ml. Immediately after a 10-min incubation period, 0.6 ml of sample headspace was analyzed for N20 as described in the Method section. The headspace of all samples wcre flushed with a stream of nitrogen for a minimum of 5 rain to remove unreacted NO before analysis of remaining AIDH thiols. *After incubation, the AIDH sulfhydryl content was measured spectrophotometrically at 412 nm using 0.5 txmol EIIman's reagent added directly to the reaction mix (16). "~Similarly treated controls containing DEA/NO, but no AIDH, produced detectable but <0.5 nmol N20. whereas N20 was undetectable in samples without DEA/NO. The amount of NzO reported was not corrected for this baseline level of N20, and therefore the ratio of SH oxidized to N20 produced is slightly nnderreportcd.



residues of adjacent subunits with the latter crosslinking these subunits. Electrophoretic patterns from nonreducing SDSP A G E carried out with NO-inhibited A1DH and similarly treated enzyme without DEA/NO were identical, that is, showed a single major band equivalent to a molecular weight of about 60,000 with no detectable bands present above this molecular weight (data not shown). These results indicate that inhibition of A1DH by NO was limited to intrasubunit disulfide bond formation. Because disulfide bond formation may cause conformational changes at the active site, the capacity of the NO-inhibited enzyme to bind NAD + and N A D H may be lost. The binding of N A D H to active and NO-inhibited A1DH was compared by measuring the fluorescence emission spectra of N A D H in the absence and presence of enzyme (5). The active and NOinhibited enzyme enhanced NADH fluorescence similarly (i.e., by 15.9 _+ 2.0% and 11.5 _+ 1.0%, respectively), which was accompanied by a downward shift in the fluorescence peak from 460 to 458 nm (data not shown). DISCUSSION Our results show that NO is a potent inhibitor of A1DH. The addition of NO, either as a dissolved gas in buffer or generated in situ from DEA/NO, inhibited the enzyme in a time- and concentration-dependent manner (Figs. 1 and 3). The enhancement of inhibition by NAD + (Fig. 2) and NADP +, but not by N A D H or NADPH, and the blockade of inhibition by the substrate, benzaldehyde (Fig. 4), is consistent with an interaction between NO and the active site cysteine as well as the ordered reaction mechanism for this enzyme (14). The inhibitor constant for NO could not be determined because the NO concentration in solution rapidly decreases over time. For example, after the addition of gaseous NO in solution to the reaction mixture at 37°C (Fig. 1), the added NO rapidly diffuses into the headspace of the reaction vessel (2). With DEA/NO, NO is slowly generated in solution according to first-order kinetics (25) and is simultaneously lost by diffusion to the headspace. In the presence of oxygen, NO would also be removed from solution by oxidation to higher oxides of nitrogen. Therefore, under both experimental protocols, the actual NO concentration in solution peaks early in the incubation period and then decreases over time. The ICs0values for inhibition of yeast AIDH by gaseous NO and DEA/NO were approximately 40 txM when determined under anaerobic conditions. The actual NO concentration in solution required to effect 50% inhibition is much lower than 40 IxM for the reasons given above. For comparison, the maximum physiologically relevant NO concentrations are estimated to be in the range of 0.45 to 10 IxM (51). The inactivation of AIDH must be considered in terms of NO concentration × exposure time. Whereas the NO concentration in solution in our studies may have been equal or even briefly higher than maximal physiologically relevant levels, the NO exposure time was limited to seconds or minutes in contrast to the continuous exposure of the enzyme to steady-state levels of NO in vivo. Yeast and mammalian A1DH isozymes contain juxtapositioned sulfhydryl groups at their active centers (19,49) that can form intramolecular disulfide bonds. In contrast, G A P D H contains active site Cys-149, which does not form an intramo|ecular disulfide. The latter is evidenced by the formation of relatively stable sulfenic acid and thionitrite forms of the active site Cys-149 (3,27,37). If a proximal sulfhydryl group were located at the active site of GAPDH, the remaining sulfhydryl

group would have readily reacted with either the sulfenic acid or the thionitrite forms of Cys-149 to produce an intramolecular disulfide (1,8). Two possible inhibitory species present in NO-containing solutions are NO itself and N203 (24). The reaction of a sulfhydryl group with NO yields a sulfenic acid [eq. (2)], which readily reacts with a second sulfhydryl to give a disulfide [eq. (3)] (13). The reaction of N203 with R-SH + 2 NO--~R-SOH + N20


R-SOH + R'-SH--~R-SS-R' + H20


a sulfhydryl group yields a thionitrite [eq. (4)] (7), In turn, thionitrites transnitrosate free sulfhydryls groups [eq. (5)], react with mercaptans to form disulfides [eq. (6)], or slowly decompose to disulfides and NO [eq. (7)] (7,36). Thus, inhibition of A1DH by NO N20 ~ + R-SH--~R-SNO + NO 2 + H + R-SNO + R'-SH--~R-SH + R'-SNO

(4) (5)

R-SNO + R'-SH--~R-SS-R' + HNO (nitroxyl)


2 R-SNO--~R-SS-R + 2 NO


and by N203 can produce the same form of the inhibited enzyme (i.e., a protein disulfide). Although S-nitrosylation and NO oxidation of the sulfhydryl groups on AIDH may lead to protein disulfide formation, their reaction intermediates are quite different. Because the occurrence of N20 ~ in NO solutions is dependent on the presence of oxygen, the relative contribution of N20~ to the overall inhibition of A1DH by NO was assessed using aerobic and anaerobic conditions. The concentration-inhibition curves (Fig. 3) for the inhibitory effect of gaseous NO and DEA/NO on AIDH activity under aerobic conditions were significantly shifted to the right, indicating that N203 is not a major contributor to the overall inhibition of A1DH by these agents. Our proposed mechanism for the inhibition of AIDH by NO is shown in Scheme I. According to this mechanism, two molecules of NO are required for the oxidation of one cysteinc residue to an S-(N-nitroso)hydroxylamino intermediate. This intermediate then undergoes a proton-assisted elimination of hydroxide ion to give a thiiranium ion intermediate, which may then undergo solvolytic disproportionation to a sulfenic acid and N20 (path a) followed by nucleophilic attack by a proximal cysteine sulfhydryl to give the intramolecular disulfide. Alternatively, the thiiranium ion intermediate may undergo intramolecular nucleophilic attack by the proximal sulfhydryl group of the enzyme (path b), thereby bypassing the sulfenic acid. We assume that NO initially reacts with Cys-302, whereas the second cysteine residue involved in this mechanism may be one proximal to Cys-302 as a result of threedimensional folding of the protein structure. Both pathways lead to the formation of an intramolecular disulfide and N20. The NO-inhibited AIDH (i.e., the disulfide form of the enzyme) was readily reactivated by DTT, but not by GSH (Table 2) or mercaptoethanol. This difference occurs because the reaction between GSH and a protein disulfide yields protein-(SH) (SSG) with the free sulfhydryl in close proximity to the mixed disulfide. This renders the reverse reaction back to the original protein disulfide much more favorable than the reduction of the intermediate to protein-(SH)2 and GSSG by a second GSH molecule (9). In contrast, the mixed disulfide formed by the reaction of DTY with a protein disulfide can be reduced by the proximal free sulfhydryl group of DTT itself giving oxidized DTF (a cyclic disulfide) and the reduced protein-(SH)2.



The reaction of D E A / N O derived NO with the vicinal dithiol DTI', a chemical model for A1DH, produced N20 as expected (Table 3). When DTT was limiting and D E A / N O was in excess, the ratio of sulfhydryl oxidized to N20 produced in this reaction had previously been shown to be 2:1 (13). This ratio agrees with the stoichiometry of 1.9 found for the reaction of NO with the sulfhydryl groups on A I D H (Table 4) and is predicted according to our mechanism (Scheme I). Because a thionitrite derivative of the active site cysteine was initially considered a possible form of the NO-inhibited AIDH, and D T I ~readily reactivated the inhibited enzyme, the reaction of DTT with GS-NO was examined. DTT reduced GS-NO to hydroxylamine (Table 2) according to Scheme II, presumably through a nitroxyl intermediate as depicted. The reduction of a thionitrite by DTT to hydroxylamine may be useful for studying other thiol containing enzymes inhibited by N203 or GS-NO. D T F had little effect on SULFI/NO derived N20 (Table 3), thus confirming a previous observation that nitroxyl is not an intermediate in the formation of N20 from SULFI/NO with the latter decomposing directly to hyponitrous acid and SO 3 (38,45). Of mechanistic significance, N20 production during inhibition of A I D H by NO also occurs without the intermediacy of nitroxyl (Scheme I). Yeast A1DH has eight easily modified thiols, presumably two per subunit (44). From the data given in Table 4, the sulfhydryl content of untreated A1DH was calculated to be 7.5 tool sulfhydryl per mol of enzyme vs. 3.9 mol sulfhydryl per mol enzyme for the NO-treated enzyme. The difference, 3.6 mol, represents the amount of A1DH sulthydryl oxidized by NO. Because the ratio of sulfhydryl oxidized to N20 produced was nearly 2, we conclude that the inhibition of enzyme activity by NO was the result of intramolecular disulfide bond formation, as depicted in Scheme I. These results also show that only half of the total titratable (Ellman's reagent) A I D H sulfhydryls was oxidized by NO even though complete inhibition of A1DH had occurred. In contrast, Ellman's reagent itself causes a parallel loss of enzyme activity and sulfhydryl content until all eight sulfhydryl groups have been modified (5). Our NO data and the nonreducing SDS-Page analyses suggest that only two of the four subunits of A I D H are catalytically active and the sulfhydryl groups of the catalytically active subunits are preferentially

/Enz'SH" J

.,SH +

Active AIDH

= Radical Intermediate

CoH Enz.;H "N=O S-(N-Nitroso)hydroxylamino Intermediate Path a H* q Path b



H20 EnZ,,sS~HN~. ~

"'°;,. L aH OH


En{,~: H + Sulfenic Acid

1 l



1 Enz'.!

N20 Nitrous

+ N20 Nitrous Oxide Intrarnolecular Oxide Disulfide



enz/,Z + H20

Inhibited AIDH)

Intramolecular Disulfide

(Reversibly Inhibited

AIDH) SCHEME I. Mechanism for the inactivation of AIDH by NO based inhibitors.

GS-N---O +




"Csx, o. --5-S~k~OH




H,, _







SCHEME II. Reduction of GS-NO to hydroxylamine by reduced DTT.



o x i d i z e d by N O to f o r m i n t r a s u b u n i t disulfide b o n d s . T h e s e f i n d i n g s are c o n s i s t e n t with " h a l f - o f - t h e - s i t e ' " r e a c t i v i t y for the t e t r a m e r i c yeast and m a m m a l i a n isozymes o f A 1 D H (5,46). In s u m m a r y , t h e m e c h a n i s m for t h e i n h i b i t i o n o f A I D H by N O has b e e n s h o w n to involve the oxidation o f proximal active site c y s t e i n e thiols to give an i n t r a s u b u n i t disulfide a n d N 2 0 . T h e p r e s e n c e o f o x y g e n significantly r e d u c e d t h e e x t e n t o f inhibition o f A 1 D H by N O , suggesting that N 2 0 ~ is not a m a j o r c o n t r i b u t o r to A I D H inhibition u n d e r physiological conditions.


ACKNOWLED(}EMEN~I S The authors wish to thank Dr. David J. W. Goon for synthesizing Angeli's salt, Yul Yost for preparing isobutyraldehyde oxime, Dr. Larry Keefer. NCI, NIH, for providing the D E A / N O and SULFI/ NO, Daniel P, Nelson for the measurements of NO by chemiluminescence, and Richard R. Erickson for nonreducing SDS-PAGE analyses. The NO Chemiluminescence Analyzer facilities were generously provided by Dr. Stephen L. Archer. This work was supported by thc Department of Veterans Affairs.


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