Báo cáo khoa học: Reduction of S-nitrosoglutathione by human alcohol dehydrogenase 3 is an irreversible reaction as analysed by electrospray mass spectrometry pptx

Reduction of S -nitrosoglutathione by human alcohol dehydrogenase 3is an irreversible reaction as analysed by electrospray mass spectrometry From the Department of Medical Biochemistry a

Reduction of S -nitrosoglutathione by human alcohol dehydrogenase 3

is an irreversible reaction as analysed by electrospray mass

spectrometry

From the Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

Human alcohol dehydrogenase 3/glutathione-dependent

formaldehyde dehydrogenase was shown to rapidly and

irreversibly catalyse the reductive breakdown of

S-nitroso-glutathione The steady-state kinetics of

S-nitrosogluta-thione reduction was studied for the wild-type and two

mutated forms of human alcohol dehydrogenase 3,

muta-tions that have previously been shown to affect the oxidative

efficiency for the substrate S-hydroxymethylglutathione

Wild-type enzyme readily reduces S-nitrosoglutathione with

S-hydroxy-methylglutathione oxidation, resulting in the highest

cata-lytic efficiency yet identified for a human alcohol

dehydrogenase In a similar manner as for

S-hydroxy-methylglutathione oxidation, the catalytic efficiency of

S-nitrosoglutathione reduction was significantly decreased

by replacement of Arg115 by Ser or Lys, supporting similar

substrate binding NADH was by far a better coenzyme than

NADPH, something that previously has been suggested to prevent reductive reactions catalysed by alcohol

However, the major products of S-nitrosoglutathione reduction were identified by electrospray tandem mass spectrometry as glutathione sulfinamide and oxidized glutathione neither of which, in their purified form, served as substrate or inhibitor for the enzyme Hence, the reaction products are not substrates for alcohol dehydrogenase 3 and the overall reaction is therefore irreversible We propose that alcohol dehydrogenase 3 catalysed S-nitrosoglutathione reduction is of physiological relevance in the metabolism of

NO in humans

Keywords: alcohol dehydrogenase; glutathione-dependent formaldehyde dehydrogenase; mass spectrometry; nitric oxide; S-nitrosoglutathione

The biological action of nitric oxide (NO) includes

vaso-dilation, inhibition of platelet aggregation and

neurotrans-mission [1] In addition to the endogenous production, NO

is also a common air pollutant, a component of cigarette

smoke and is generated during metabolism of several

pharmaceutical drugs [2,3] It has been suggested that many

intracellular processes of NO involve nitrosylation of thiols

[4] S-nitrosothiols have been proposed to affect ventilation

[5], alter protein function [4], act as bioactivators of nitrites

and nitrates [6] or serve as a
pool of NO [7] Recently,

attention has been drawn to the S-nitrosothiol of

glutathi-one (GSH), i.e S-nitrosoglutathiglutathi-one (GSNO) where GSH

may alternatively act as a scavenger for NO to withstand

nitrosative stress [8] or act as a modulator of the action of

NO [9] GSNO has been proposed to be produced under physiologically relevant conditions [10–12] and indeed, it has been detected in various biological systems including rat cerebellum and primary glial cell cultures [8,9] as well as in human airways [13]

As GSNO potentially has significant roles in various biological processes, its fate and breakdown is of consider-able interest In addition to its complex chemical fate [14,15], the enzymatic reduction of GSNO by rat alcohol dehy-drogenase 3 (ADH3), also known as GSH-dependent formaldehyde dehydrogenase, has been investigated [16] More recently studies on ADH3–/– mice and yeast have demonstrated that ADH3 is important for GSNO metabo-lism and may regulate intracellular S-nitrosothiol levels in these model systems [17] Furthermore, plant ADH3 also possesses GSNO reductase activity indicating the activity to

be general [18] It is noteworthy with respect to ADH activities, that reductive reactions are generally not believed

to be of physiological relevance due to the low NADH/

also applicable to the metabolism of GSNO by ADH3 ADH3 belongs to the medium chain alcohol dehydro-genase (ADH) system which, according to current nomen-clature, is divided into five classes in man ADH1–ADH5 [21] in which the ADH proteins and genes have been designated the same number All ADHs display oxidative/ reductive enzymatic activities for a variety of alcohols/ aldehydes of both endogenous and exogenous origin [22,23]

Correspondence to J.-O Ho¨o¨g, Department of Medical Biochemistry

and Biophysics, Karolinska Institutet, SE-171 77 Stockholm,

Sweden Fax: +468 338453, Tel.: +468 728 7740,

E-mail: jan-olov.hoog@mbb.ki.se

Abbreviations: ADH, alcohol dehydrogenase; ADH3, alcohol

dehy-drogenase 3/GSH-dependent formaldehyde dehydehy-drogenase; GSNO,

S-nitrosoglutathione; HMGSH, S-hydroxymethylglutathione;

NO, nitric oxide.

*Present address: Amersham Biosciences, SE-751 84 Uppsala, Sweden.

Present address: Department of Medical Biochemistry and

Micro-biology, Uppsala University, SE-751 23 Uppsala, Sweden.

(Received 4 November 2002, revised 17 January 2003,

accepted 27 January 2003)

oxidation and reduction of substrates, respectively Most

likely, one physiological substrate for human ADH3 is the

spontaneously formed complex of formaldehyde and GSH,

S-hydroxymethylglutathione (HMGSH) On the basis that

the enzyme is considered to be the prime guardian against

formaldehyde [24–26], shows extensive evolutionary

con-servation [27] and is expressed in all tissues examined

[28,29], ADH3 is regarded as essential for basic cell

metabolism possibly also including retinoic acid production

during growth [30]

GSNO is a recent addition to the ADH3 substrate

repertoire and here we investigate the enzymatic activity of

the human form of ADH3 for this compound

Recombi-nant expression and purification of wild-type and mutated

forms of the enzyme enabled kinetic characterization and

investigation of substrate–enzyme interactions Electrospray

tandem MS (MS/MS) was used to identify the products of

the enzymatic reduction of GSNO, which were previously in

doubt Finally, we conclude that the glutathione sulfinamide

formed is not a substrate for ADH3, thus ADH3 catalysed

GSNO reduction is irreversible

Materials and methods

Enzyme purification and chemicals

Generation of expression vectors for recombinant

expres-sion of the wild-type and mutant forms of ADH3 have

previously been described [31] The various forms of ADH3

were expressed in Escherichia coli and purified to

homo-geneity in a three-step procedure essentially as described

[31] Protein concentrations were determined

colourimetri-cally [32] and the purity was analysed by SDS/PAGE

were purchased from Boehringer Mannheim GSH, GSSG,

NADPH and GSNO were from Sigma Formaldehyde

solutions in acetonitrile were made from newly opened glass

ampoules, 20% solutions (Ladd Research Industries)

Kinetic analysis

Enzymatic activity was monitored by following the

absorb-ance change at 340 nm with a Hitachi U-3000

NADH reduction/oxidation the additive molar absortivity

HMGSH concentrations were calculated according to [33]

Deter-mination of kinetic constants for NADH and NADPH

phos-phate pH 7.5 To fit lines to data points a weighted

ADH3–Arg115Ser and GSNO reduction using NADPH as coenzyme Under these conditions the enzymes could not

be saturated and kinetic constant determinations should be regarded with caution For mass spectrometric analyses,

bicarbonate pH 7.5, as phosphate ions severely impair mass spectrometric analysis Reaction velocities were not signifi-cantly different in this buffer as compared to that used for kinetic analyses

MS Electrospray mass and tandem mass spectra were recorded

on an AutoSpec-OATOFFPD high-resolution magnetic sector-orthogonal acceleration time-of-flight (OATOF) tan-dem mass spectrometer (Micromass) Both positive- and negative-ion nano-electrospray spectra were recorded by spraying sample mixtures from metal coated borosilicated capillaries Tandem mass spectra were recorded by selecting the desired precursor ion with the double focussing sectors

of the instrument (MS1) focussing them into an inter-mediate collision cell containing Xe and orthogonally accelerating undissociated precursors and fragment ions into the time-of-flight (TOF) analyser (MS2)

Product isolation

In an effort to isolate the major product, i.e glutathione

for 2 h to reach equilibrium The resulting reaction products were resolved on a strong anion exchange column Table 1 Steady state kinetic constants for HMGSH/GSNO oxidation/reduction at pH 7.5, catalysed by wild-type and mutant forms of ADH3.

K m - and k cat -values were determined from initial velocity experiments at 25 C with substrate concentrations varied over a 10-fold range Values are given as means (± SE) of three independent experiments NAD + and NADH concentrations were fixed at 2.4 m M and 0.1 m M , respectively.

Enzyme

K m

(l M )

k cat

(min)1)

k cat /K m

(min)1Æm M )1 )

K m

(l M )

k cat

(min)1)

k cat /K m

(min)1Æm M )1 )

a Values were determined under nonsaturating conditions.

(Resource Q, 1 mL; Amersham Biosciences) with a linear

an A¨KTA HPLC system (Amersham Biosciences) The

elution profile was monitored at 214 nm The identity of

the isolated products were confirmed by MS as described

above The concentration of the collected glutathione

sulfinamide was determined spectrophotometrically at

214 nm using standardized GSH solutions as references

Results

Steady-state kinetics

Steady-state kinetic constants for HMGSH, GSNO,

mutated forms of ADH3 (Tables 1 and 2) In addition,

deter-mined for the wild-type enzyme (Table 2) Human ADH3

was found to readily reduce GSNO with NADH as

Reduc-tive capacity for GSNO was drastically reduced by the

substitution of Arg115 for Ser or Lys The lowered catalytic

for the Arg115Ser and Arg115Lys mutants, respectively

in the micromolar range The catalytic capacity for

and NADPH as coenzymes were two to three orders of

coenzymes For the two mutants, kinetic constants for

NADPH (data not shown) GSSG, GSH or

not shown)

Electrospray MS/MS and isolation of the products

To determine the structure of the product generated by

ADH3 catalysed reduction of GSNO, nano-electrospray

mass spectra of the reaction mixture with and without the

addition of enzyme were recorded (Fig 1) In addition, the

same experiment was performed with and without GSH

This allowed the source of contaminant ions to be identified

(data not shown) After an incubation period of 1 h

following addition of enzyme, the peaks corresponding to

found to decrease in intensity, and new peaks at m/z 339 and

708 to appear In the reaction mixtures containing GSH, the

m/z 613 corresponded to protonated GSSG As GSH levels

in the initial reaction mixture were increased the peak

corresponding to protonated GSSG increased in intensity

When a 15-fold excess of GSH was applied, the peak at m/z

339 and the peak corresponding to protonated GSSG were

approximately equal in intensity In addition, a

correspond-ing reaction mixture without enzyme was analysed yieldcorrespond-ing

similar results with respect to GSSG formation The identity

+ ,

Km

kca

1 )

kcat

1 Æm

1 )

1 )

kcat

1 Æm

1 )

1 )

kcat

1 Æm

1 )

1 )

kcat

1 Æm

1 )

determined by MS/MS The MS/MS spectra of the peaks at

The spectra show many similarities and are characteristic of

glutathione conjugates (Fig 2 [34]) However, the spectra

differ in that the ion at m/z 307, specific for the glutathione conjugation moiety is absent in MS/MS spectrum of the ion

at m/z 339 In its place a new fragment at m/z 322 is observed

Fig 2 Mass fragmentation analyses of substrate and products obtained from the enzymatic reaction Tandem mass spectrometric fragmentation spectra of [GSNO]H+m/z 337 (upper), and protonated major product m/z 339 (lower) The schematic structures are depicted to the right with some fragmentation breaks For a more detailed description see Yang et al [34].

Fig 1 Nano-electrospray mass spectra of reaction mixtures The upper spectrum shows components without the addition of enzyme (background) and the lower spectrum shows components after the addition of ADH3 Peaks corresponding to NAD+, [NADH]H+, [GSi]H+, [GSNO]H+, [GSSG]H + and the major product are indicated The peaks at m/z 359 and 361 correspond to Na + adducts of GSNO and the
major product, those at m/z 666/688 and 664/686 are associated with NADH and NAD + , respectively, while those at m/z 279, 280 and 316 are small contaminants Peaks at m/z from 524 to 587 detected in the mass spectra with enzyme are also present in the spectra without enzyme although at much lower intensities.

In an effort to understand the fragmentation spectrum of

the ion at m/z 339, reference spectra were recorded of

protonated glutathione sulfinic acid (generated through

acidification of the reaction mixture after incubation as

described [16]), glutathione sulfonic acid and HMGSH

(Fig 3) From the spectra of these glutathione conjugates it

is evident that oxidation of the glutathione sulfur effects

fragmentation in such a way that the ion at m/z 307 is no

longer observed in the MS/MS spectrum This, along with a

comparison with reference spectra of other glutathione

conjugates recorded on this instrument [34], demonstrates

that the ion at m/z 339 has the structure of a glutathione

sulfinamide (Scheme 1)

The glutathione sulfinamide was purified using an anion

exchange column (Fig 4A) and the identity and purity was

confirmed by MS (Fig 4B) In addition to the peak

corresponding to glutathione sulfinamide additional peaks

and GSSG were resolved Notably, glutathione sulfinic acid,

and 710, respectively) were not found to be present in the

glutathione sulfinamide fraction, i.e peak 3 in Fig 4A The

glutathione sulfinamide was not a substrate for ADH3 using

oxidation by ADH3 to any extent at concentrations up to

glutathione sulfinic acid was detected (Fig 4) However, this

compound was never observed during the initial reaction

analysis as described above Probably this compound is

spontaneously formed during the exceptionally long

incu-bation used to reach equilibrium The formation of this

glutathione sulfinic acid can be enhanced by addition of acid

as described [16]

Discussion

Human ADH3 readily catalyses the reduction of GSNO

The two glutathione conjugates HMGSH and GSNO are

by far the best substrates identified for ADH3 Further, the

catalytic efficiency for GSNO reduction is almost twofold

higher than for HMGSH oxidation with the result that GSNO is the best substrate for the human ADH3 yet identified Notably, GSNO concentrations have been reported to reach micromolar levels under certain circum-stances [8,13] These observations, together with the fact that ADH3 is expressed in all tissues, lend support to the notion that ADH3 may serve as a GSNO metabolizer in humans

It has been suggested however, that due to the
normally

reactions exerted by ADHs are not favourable [20] Analysis of the major product by electrospray tandem

MS demonstrated that in addition to GSSG, the other

Fig 3 Tandem mass spectrometric fragmentation spectra of protonated glutathione sulfinic acid m/z 340 (upper) and protonated HMGSH m/z 338 (lower) The schematic structures are depicted to the right with some fragmentation breaks For a more detailed description see Yang et al [34].

Scheme 1 Transformation of GSNO to glutathione sulfinamide by ADH3.

major product is glutathione sulfinamide (Scheme 1) This

finding is in line with the previously suggested but not

confirmed structure of Jensen and coworkers [16] The

isolated glutathione sulfinamide did not serve as substrate or

inhibitor for ADH3 Other sulfinamides have been shown to

be formed spontaneously by rearrangement of the

corres-ponding semimercaptale [35], a mechanism where the S¼O

oxygen originates from the solvent It is most likely that

the isolated glutathione sulfinamide is also formed via a

semimercaptale intermediate (Scheme 1) Hence, the

over-all reduction of GSNO catalysed by ADH3 is clearly an

irreversible reaction With these observations taken into

account, the reaction may therefore be forced in the

reductive direction thereby
overriding the influence of a

to the physiological relevance

ratio is significantly changed For instance, during ethanol

intake increased levels of enzyme coenzyme complexes in rat

hepatocytes have been observed [36] and ethanol

consump-tion has also been shown to increase the output of lactate

kidney [37] It is possible that such a change may also occur

in other tissues, i.e where GSNO is produced, and thereby

influence its metabolism Naturally, such a change in redox potential would presumably also influence HMGSH meta-bolism Similar reasoning has included other ADHs and their metabolism of various endogenous substrates, e.g certain serotonin metabolites, during ethanol intake [23] The observation of GSSG as a product is in line with findings of previous investigators However, for the bacter-ial ADH3, Liu et al do not report any detection of glutathione sulfinamide but propose that the intermediate semimercaptale reacts with NADH to form glutathione amine, which is subsequently oxidized to GSSG by an additional GSH [17] We do not detect any glutathione amine for the human ADH3-catalysed reaction Moreover, Jensen et al proposes that for the mammalian enzyme, GSSG is only a minor product [16] We find that when excess GSH is added, approximately equal amounts of GSSG and glutathione sulfinamide are formed, and so both products are probably physiologically relevant Of note is that GSSG is one of the major products spontaneously formed from GSH and GSNO [14] and so it is conceivable that the GSSG formed is not generated from the enzyme catalysed reaction but rather is formed spontaneously during the incubation This was also confirmed in a control experiment without enzyme

Fig 4 Isolation and analysis of reaction products (A) After incubating the ADH3-catalysed GSNO reduction for 2 h at 37 C (see Materials and methods), the components were resolved on a strong anion exchange column (Resource Q) using 10 m M ammo-nium bicarbonate pH 7.5, 20% acetonitrile as the initial solvent and 1 M ammonium bicar-bonate pH 7.5, 20% acetonitrile as the elution solvent (buffer B) The elution positions (1) NAD + (2) NADH (3) glutathione sulfina-mide (4) glutathione sulfinic acid and (5) GSSG are indicated by arrows (B) MS of the fraction corresponding to peak 3 in (A) dem-onstrating the identity and purity of the glutathione sulfinamide Notably, no con-taminants of GSSG, GSNO, NAD + or NADH (m/z 613, 337, 708 and 710, respect-ively) were detected.

The effect of substituting Arg115 by Ser or Lys in ADH3

has previously been investigated [31] For HMGSH, an

exact positioning in the active site is essential for efficient

catalysis The substrate binding is accomplished in part by a

charge interaction between Arg115 and the glycine

carb-oxylate group in the GSH moiety However, both mutants

studied stress the requirement for exact positioning of

interacting groups in the substrate and the polypeptide

chain Like the results for HMGSH, GSNO reduction was

impaired by the replacement of Arg115 by both Ser and Lys

GSNO and HMGSH bind in similar, if not identical,

manners in the ADH3 active site cleft Moreover, HMGSH,

GSNO, GSH and methylglutathione show extensive

struc-tural similarities Still, GSH or methylglutathione did not

inhibit the enzyme, which further illustrates the exactness

with which the HMGSH or GSNO molecule interacts with

the substrate pocket

Addition of millimolar concentrations of NADPH to

cytosol fractions of rat liver has been reported to increase

the rate of GSNO disappearance [38] NADPH could

indeed be utilized as coenzyme, but with three orders of

compared to NADH (Table 2) NADPH concentrations

hepatocyte cytosol and even lower in other tissues, including

heart and brain [39], indicating that NADPH is unlikely to

be utilized in vivo by ADH3 as coenzyme for GSNO

reduction

In conclusion, human ADH3 shows high catalytic

capacity for GSNO reduction with NADH as coenzyme

Mutational analysis demonstrates that GSNO binds in a

similar manner to HMGSH to ADH3 As the major

products formed are GSSG and glutathione sulfinamide,

neither of which function as substrate or inhibitor for the

enzyme, it is now clear that the overall reaction is

irreversible We propose that human ADH3 may serve as

a GSNO metabolizer in vivo

Acknowledgements

We thank B Agerberth and M Tollin for the use of equipment and

valuable help with HPLC This work was supported by the Alcohol

Research Council of the Swedish Alcohol Retailing Monopoly, the

Swedish Match and Karolinska Institutet.

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