Biochimie (1992) 74, 557-560 557
© Soci6t~ fran~aise de biochimie et biologie mol6culaire / Elsevier, Paris
Essential arginines in mercuric reductase isolated from Yersinia enterocolitica 138A14
M B l a g h e n l , M S E l K e b b a j 1, D J M V i d o n 2, D T d t s c h 3 *
tLaboratoire de Biochimie, Facult6 des Sciences I Universit6 Hassan II, Km 8 route E! Jadida, Maarif, BP 5366, Casablanca, Morocco;
ZLaboratoire de Bact~riologie et Cryptogamie, Facultd de Pharmacie, Universit# Louis Pasteur, 74, route du Rhin, 67401 Ilikirch Cedex;
3Laboratoire de Chimie Organique Bioiogique, lnstitut de Chimie, Universit~ Louis Pasteur, 1, rue Blaise Pascal, 67008 Strasbourg Cedex, France
(Received 19 December 1991; accepted 5 March 1992)
Summary - - The mercuric reductase from Yersinia enterocolitica 138A14 was inactivated by the arginine modifying reagents 2,3- butanedione and phenylglyoxal. The inactivation by 2,3-butanedione exhibited second order kinetics with rate constant of 32 mitt -1 M -1. In the case of phenylglyoxal, biphasic kinetics were observed. The oxidized coenzyme (NADP +) prevented inactivation of the enzyme by the ct-dicaxbonyl reagents, whereas the reduced coenzyme (NADPH) enhanced the inactivation rate. The loss of enzyme activity was related to the incorporation of [2-t4C] phenylglyoxal; when two arginines per subunit were modified the enzyme was completely inactivated.
mercuric reductase / phenylglyoxal / 2,3-butanedione / arginine modification
Introduction
Mercuric reductase plays a crucial role in bacterial detoxification o f mercurials [1-3]. The enzyme cata- lyzes the two-electron reduction of mercuric ions to elemental mercury with concomitant oxidation of NADPH. The presence of exogenous thiol-compounds is necessary for enzymatic activity. The enzyme contains a FAD and a redox-active disulfide (Cyst36- Cys140 (the numbering of amino acids corresponds to the sequence o f T n S O l mercuric reductase [4] whose HgR region o f the transposon displays homology with those of T n 3 9 2 6 , the mercury resistance transposon of Yersinia [5]) at the active site [1, 3, 6]. Another thiol pair, Cyssss-Cys559 plays a essential role in binding and positioning mercury ions for reduction [71. An hypothetical catalysis mechanism was proposed for the dimeric T n S O l mercuric reductase [8].
In order to get further information on the active site amino acids, we studied the effect o f phenylglyoxal and 2,3-butanedione towards mercuric reductase from Fersinia e n t e r o c o l i t i c a 138A14. As E s c h e r i c h i a coli R831 mercuric reductase [9], the enzyme from Yersi- nia enterocolitica 138A14 seems to be a trimer of
*Correspondence and reprints
200 kDa (subunit molecular mass 70 kDa). During storage, proteolytic degradation leads to the formation o f dimeric molecules with a molecular weight of 105 kDa (subunit 52 kDa) [10]. In this report we show that the two site-specific reagents inactivated the enzyme, suggesting the presence of arginyl residues in the active site. The localization of these arginines is discussed according to the three-dimensional struc- tures of mercuric reductase isolated from Bacillus sp strain RC607 [11] and of human glutathione reductase which exhibits extensive similarities [ 11,12].
Materials and methods Purification of the enzyme
Mercuric reductase was isolated from Yersinia enterocolitica 138A14 by the method described elsewhere [10]. After the elu- tion from the Cibacmn blue Matrex column, enzyme-bound NADP* was removed via exhaustive dialysis of the enzyme against a solution of KBr (2 M) in a 30 mM phosphate buffer, pH 7.5 [1]. The mercuric reductase (> 90%) was in the native non-proteolyzed form.
Determination of the enzymatic activity.
Assays were performed at 340 nm and 25°C in a 50 mM phos-
phate buffer (pH 7.5) containing 0.1 mM NADPH, 0.3 mM
558
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10 0 10 20 30
T i m e ( m i n )
Fig I. Inactivation of mercuric reductase by 2,3-butane- dione. The enzyme (0.46 mg/ml) was inactivated by 2,3- butane-dione 1 mM (=), 1.5 mM (e), 2 m M ( = ) , 3 mM ( o ) at 25°C in borate buffer (25 mM) pH 8. The inset shows the plot of the inactivation rate (kob,) versus the concentration of 2,3-butanedione.
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m m =1
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Fig 2. Inactivation of mercuric reductase by phenylglyoxal.
The enzyme (0.46 mg/ml) was inactivated by phenylglyoxal 4 mM (-), 5 mM (e), I0 mM C=) and 15 mM ( o ) at 25°C in phosphate buffer (30 raM) pH 7.5. The inset shows the plot of the inactivation rate (ko~) versus the concentration of phenylglyoxal.
HgC! 2 and 5 mM L-cysteine. One unit of enzymatic activity is defined as the amount of enzyme which catalyzes the oxidation of 1 I~mol of NADPH per minute under assay conditions.
Protein concentration
The protein concentration was measured by the Bradford method using bovine serum albumin as the standard 113]. The enzyme concentration was calculated using a molecular mass/subunit of 70 kDa.
Modification of mercuric reductase with phenyiglyoxal and 2,3-butanedione
Purified enzyme (0.46 mg/ml) was incubated at 25°C with phenyl glyoxal (4--15 mM final concentration) in 30 mM phos- phate buffer pH 7.5. At fixed times, aliquots were withdrawn and the residual activity was measured. Chemical modification with 2,3 batanedione (1.5-4 mM final concentration) was performed in 25 mM borate buffer, pH 8. Control assays were performed under the same conditions except that the modifying reagents were omitted. The influence of NADP + (1 mM) and NADPH (0.05 mM) on the inactivation rate were determined.
To determine the influence of the disulfide opening in the presence of NADPH on the inactivation rate, mercuric reductase was preincubated with 1 mM NADPH. The flee thiol groups were reversibly blocked by reaction with 0.1 mM 5,5'- dithiobis(2-nitrobenzoic acid). Phenylglyoxal (15 raM) was then added to the reaction medium and the residual activity was determined as described above. The free thiols of the enzyme were regenerated by the cysteine present in the assay medium.
Incorporation of 12-14C] phenylglyoxal into the enzyme [2-]4C] Phenyiglyoxal (specific activity: 27 mCi/mmol) was purchased from CEA (Saclay, France) and diluted ten-fold with phenylglyoxal. Mercuric reductase (0.46 mg/ml) was modified with 5 mM [2-14C] phenylglyoxal. Incorporation of 12-14C]
phenyiglyoxal into mercuric reductase was assayed as acid- insoluble radioactivity by the paper-disc method of Bollum [14]. Blanks were determined using the same conditions but in the presence of NADP + (1 raM). Radioactivity was measured using an Intertechnique liquid scintillation spectrometer.
Results and discussion
The 0¢-dicarbonyl reagents phenylglyoxai and 2,3- butanedione are known to react with the guanidinium group of arginine. 2,3-Butanedione forms a specific, reversible complex with arginine [15, 16] which is stabilized by borate buffer. The modification of argi- nine with phenylglyoxal is irreversible and two mole- cules of reagent bind per arginine residue [17-19].
The inactivation of mercuric reductase by 2,3-buta- nedione showed pseudo first-order kinetics [20]. The second-order rate constant was 32 min-I M-I (figl).
Phenylglyoxai inactivated mercuric reductase entirely
and irreversibly. The inactivation followed biphasic-
kinetics so that more than one arginine residue
seemed to be implicated in the inactivation of the
enzyme. The first phase was very fast and could not
be analyzed. The second phase of the inactivation fol-
lowed pseudo-first order kinetics. The second-order
rate constant was 3.3 min-~ M -I (fig 2). Concerning
the inactivation profile, the difference between 2,3-
butanedione and phenyiglyoxal could be attributed to
the fact that phenylglyoxal is a bulkier molecule than
2,3-butanedione so the modification of one guanidino
group could prevent the modification of a second by
steric hindrance. Phenyiglyoxal has a rather hydro-
phobic character while 2,3-butanedione is a hydro-
philic molecule. Depending on the environment of the arginine residues, the reactivity towards the ot-di- carbonyl reagents could be quite different.
The time course o f [2-14C1 phenylglyoxal incorpor- ation into the mercuric reductase revealed that the loss of activity was linearly related to the incorporation of radioactivity. Since NADP + (1 raM) completely pro- tected the enzyme from inactivation by phenylglyoxal and 2,3-butanedione (results not shown), blanks were determined in the presence of NADP* to prevent the numbering of non-essential Arg residues. Extra- polation to zero activity correlated with the modifi- cation of about two Arg residues per subunit (fig 3).
These results suggest that mercuric reductase is com- pletely inactivated when two Arg residues, located in
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