Johan Ålander

Reduction of S-nitrosoglutathione by alcohol dehydrogenase 3 is facilitated by substrate alcohols via direct cofactor recycling and leads to GSH-controlled formation of glutathione transferase inhibitors.

GSNO (S-nitrosoglutathione) is emerging as a key regulator in NO signalling as it is in equilibrium with S-nitrosated proteins. Accordingly, it is of great interest to investigate GSNO metabolism in terms of competitive pathways and redox state. The present study explored ADH3 (alcohol dehydrogenase 3) in its dual function as GSNOR (GSNO reductase) and glutathione-dependent formaldehyde dehydrogenase. The glutathione adduct of formaldehyde, HMGSH (S-hydroxymethylglutathione), was oxidized with a k(cat)/K(m) value approx. 10 times the k(cat)/K(m) value of GSNO reduction, as determined by fluorescence spectroscopy. HMGSH oxidation in vitro was greatly accelerated in the presence of GSNO, which was concurrently reduced under cofactor recycling. Hence, considering the high cytosolic NAD(+)/NADH ratio, formaldehyde probably triggers ADH3-mediated GSNO reduction by enzyme-bound cofactor recycling and might result in a decrease in cellular S-NO (S-nitrosothiol) content in vivo. Formaldehyde exposure affected S-NO content in cultured cells with a trend towards decreased levels at concentrations of 1-5 mM, in agreement with the proposed mechanism. Product formation after GSNO reduction to the intermediate semimercaptal responded to GSH/GSNO ratios; ratios up to 2-fold allowed the spontaneous rearrangement to glutathione sulfinamide, whereas 5-fold excess of GSH favoured the interception of the intermediate to form glutathione disulfide. The sulfinamide and its hydrolysis product, glutathione sulfinic acid, inhibited GST (glutathione transferase) activity. Taken together, the findings of the present study provide indirect evidence for formaldehyde as a physiological trigger of GSNO depletion and show that GSNO reduction can result in the formation of GST inhibitors, which, however, is prevented under normal cellular redox conditions.

The Janus face of alcohol dehydrogenase 3.

Many carbonyl metabolizing enzymes are equally involved in xenobiotic and endogenous metabolism, but few have been investigated in terms of substrate competition and interference between different cellular pathways. Mammalian alcohol dehydrogenase 3 (ADH3) represents the key enzyme in the formaldehyde detoxification pathway by oxidation of S-hydroxymethylglutathione [HMGSH; the glutathione (GSH) adduct of formaldehyde]. In addition, several studies have established ADH3 as S-nitrosoglutathione (GSNO) reductase in endogenous NO homeostasis during the last decade. GSNO depletion associates with various diseases including asthma, and evidence for a causal relationship between ADH3 and asthma pathology has been put forward. In a recent study, we showed that ADH3-mediated alcohol oxidation, including HMGSH oxidation, is accelerated in presence of GSNO which is concurrently reduced under immediate cofactor recycling [C.A. Staab, J. Alander, M. Brandt, J. Lengqvist, R. Morgenstern, R.C. Grafström, J.-O. Höög, Reduction of S-nitrosoglutathione by alcohol dehydrogenase 3 is facilitated by substrate alcohols via direct cofactor recycling and leads to GSH-controlled formation of glutathione transferase inhibitors, Biochem. J. 413 (2008) 493-504]. Thus, considering the usually low cytosolic free NADH/NAD(+) ratio, formaldehyde may trigger and promote GSNO reduction by enzyme-bound cofactor recycling. These findings provided evidence for formaldehyde-induced, ADH3-mediated GSNO depletion with potential direct implications for asthma. Furthermore, analysis of product formation as a function of GSH concentrations suggested that, under conditions of oxidative stress, GSNO reduction can lead to the formation of glutathione sulfinamide and its hydrolysis product glutathione sulfinic acid, both potent inhibitors of glutathione transferase activity.

Microsomal glutathione transferase 1 exhibits one-third-of-the-sites-reactivity towards glutathione

The trimeric membrane protein microsomal glutathione transferase 1 (MGST1) possesses glutathione transferase and peroxidase activity. Previous data indicated one active site/trimer whereas structural data suggests three GSH-binding sites. Here we have determined ligand interactions of MGST1 by several techniques. Nanoelectrospray mass spectrometry of native MGST1 revealed binding of three GSH molecules/trimer and equilibrium dialysis showed three product molecules/trimer (K(d)=320+/-50 microM). All three product molecules could be competed out with GSH. Reinvestigation of GSH-binding showed one high affinity site per trimer, consistent with earlier data. Using single turnover stopped flow kinetic measurements, K(d) could be determined for a low affinity GSH-binding site (2.5+/-0.5 mM). Thus we can reconcile previous observations and show here that MGST1 contains three active sites with different affinities for GSH and that only the high affinity site is catalytically competent.

Characterization of a new fluorogenic substrate for microsomal glutathione transferase 1

A new thiol-reactive electrophilic, disubstituted rhodamine-based fluorogenic probe (bis-2,4-dinitrobenzenesulfonyl rhodamine [BDR]) with very high quantum yield was synthesized and described recently [A. Shibata et al., Bioorg. Med. Chem. Lett. 18 (2008) 2246-2249]. Because hydrophobic electrophiles are often conjugated by glutathione transferases, the BDR or monosubstituted rhodamine derivatives (2,4-dinitrobenzenesulfonyl rhodamine [DR]) were tested with microsomal glutathione transferase 1 (MGST1) and shown to function as substrates. The kinetic parameters for purified enzyme and DR were k(cat)=0.075+/-0.005 s(-1) and K(m)=21+/-3 microM (k(cat)/K(m)=3.6 x 10(3)+/-5.6 x 10(2)M(-1)s(-1)), giving a rate enhancement of 10(6) compared with the nonenzymatic reaction. In cells overexpressing MGST1, the addition of BDR caused a time-dependent increase of fluorescence compared with control cells. Preincubating the cells with a thiol reagent (N-ethylmaleimide) abolished the fluorescent signal. By using DR, we could determine the MGST1 activity in whole cell extracts with high sensitivity. In addition, the activity could be increased by thiol reagents (a hallmark of MGST1). Thus, we have identified a new fluorogenic substrate for MGST1 that will be a useful tool in the study of this enzyme and related enzymes.

Dead-end complex, lipid interactions and catalytic mechanism of microsomal glutathione transferase 1, an electron crystallography and mutagenesis investigation.

Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used electron crystallography of two-dimensional crystals in order to determine an atomic model of rat MGST1 in a lipid environment. The model comprises 123 of the 155 amino acid residues, two structured phospholipid molecules, two aliphatic chains and one glutathione (GSH) molecule. The functional unit is a homotrimer centered on the crystallographic three-fold axes of the unit cell. The GSH substrate binds in an extended conformation at the interface between two subunits of the trimer supported by new in vitro mutagenesis data. Mutation of Arginine 130 to alanine resulted in complete loss of activity consistent with a role for Arginine 130 in stabilizing the strongly nucleophilic GSH thiolate required for catalysis. Based on the new model and an electron diffraction data set from crystals soaked with trinitrobenzene, that forms a dead-end Meisenheimer complex with GSH, a difference map was calculated. The map reveals side chain movements opening a cavity that defines the second substrate site.