Mitochondrial Nicotinamide Nucleotide ... - ACS Publications

Public Health Service Grant GM24887. Publi- cation No. 5643-BCR from the Research Institute of Scripps Clinic, La. Jolla, CA. *To whom correspondence ...
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Biochemistry 1989, 28, 6050-6056

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Mitochondrial Nicotinamide Nucleotide Transhydrogenase: NADPH Binding Increases and NADP Binding Decreases the Acidity and Susceptibility to Modification of Cysteine-893+ Mutsuo Yamaguchi and Youssef Hatefi* Division of Biochemistry, Department of Basic and Clinical Research, Research Institute of Scripps Clinic, La Jolla, California 92037 Received February 27, 1989

ABSTRACT: The mitochondrial nicotinamide nucleotide transhydrogenase is a dimeric enzyme of monomer

M , 1 10 000. It is located in the inner mitochondrial membrane and catalyzes hydride ion transfer between NAD(H) and NADP(H) in a reaction that is coupled to proton translocation across the inner membrane. The amino acid sequence and the nucleotide binding sites of the enzyme have been determined [Yamaguchi, M., Hatefi, Y., Trach, K., & Hoch, J. A. (1988) J . Biol. Chem. 263,2761-2767; Wakabayashi, S., & Hatefi, Y. (1987) Biochem. Znt. 15,915-9241, N-Ethylmaleimide, as well as other sulfhydryl group modifiers, inhibits the transhydrogenase. The presence of N A D P in the incubation mixture suppressed the inhibition rate by N-ethylmaleimide, and the presence of N A D P H greatly increased it. N A D and N A D H had little or no effect. The N A D P H effect was concentration dependent and saturable, with a half-maximal N A D P H concentration effect close to the K , of the enzyme for NADPH. Study of the effect of p H on the Nethylmaleimide inhibition rate showed that N A D P H binding by the enzyme lowers the apparent pK, of group by 0.4 of a pH unit and N A D P binding raises this pK, by 0.4 of a the N-ethylmaleimide-sensitive pH unit, thus providing a rationale for the effects of NADP and NADPH on the N-ethylmaleimide inhibition rate. With the use of N - [3H]ethylmaleimide, the modified sulfhydryl group involved in the NADP(H)modulated inhibition of the transhydrogenase was identified as that belonging to Cys-893, which is located 1 13 residues upstream of the tyrosyl residue modified by [p-(fluorosulfonyl)benzoyl]-5’-adenosinea t the putative NADP(H) binding site of the enzyme (see above references). In addition, data have been presented suggesting that Cys-893 is not an essential residue and that enzyme inhibition is partial when the modifier is methyl methanethiosulfonate which introduces a smaller group (-SCH3) at the modified cysteine sulfhydryl than does N-ethylmaleimide. However, regardless of its role in catalysis, Cys-893 has provided a means to show that substrate and product binding cause opposite conformation changes in the transhydrogenase. This is the first instance that such a phenomenon, which has important mechanistic implications regarding proton pumping, has been demonstrated for any energy-transducing enzyme of the mitochondrial or bacterial oxidative phosphorylation system.

%e mitochondrial nicotinamide nucleotide transhydrogenase is a dimeric enzyme of monomer M , 110 000 (Anderson & Fisher, 1981; Yamaguchi et al., 1988a). It is located in the inner mitochondrial membrane and catalyzes the energy-driven transfer of a hydride ion from NADH to NADP according to eq 1, where H: and H i denote protons on the cytosolic and NADH

+ NADP + nH,f + NAD + NADPH + nH: (1)

the matrix sides of the inner mitochondrial membrane, respectively, and n appears to be unity (Earle & Fisher, 1980; Eytan et al., 1987). Mitochondrial membrane energization via respiration or ATP hydrolysis increases the rate of NADP reduction by more than 10-fold and shifts the equilibrium of reaction 1 toward product formation (Rydstrom, 1977; Fisher & Earle, 1982). The bovine enzyme exhibits half-of-the-sites reactivity (Phelps & Hatefi, 1984a,b, 1985), and there is kinetic and thermodynamic evidence suggesting that it undergoes cyclic conformation change during catalysis (Hatefi et al., 1980, 1982). Previous studies from this laboratory have ‘Supported by US.Public Health Service Grant GM24887. Publication No. 5643-BCR from the Research Institute of Scripps Clinic, La Jolla, CA. *To whom correspondence should be addressed.

0006-2960/89/0428-6050$0 1.50/0

elucidated the amino acid sequences of the bovine enzyme and its signal peptide (Yamaguchi et al., 1988a,b), and have identified the substrate binding sites (Wakabayashi & Hatefi, 1987a). The amino acid sequences of the two subunits of the Escherichia coli transhydrogenase have also been reported (Clarke et al., 1986). The bovine transhydrogenase is inhibited by a large number of protein modifying reagents, including thiol group modifiers (O’Neal & Fisher, 1977; Earle et al., 1978; Persson & Rydstrom, 1987), dicyclohexylcarbodiimide(DCCD) (Phelps & Hatefi, 1981, 1984a; Pennington & Fisher, 1981; Wakabayashi & Hatefi, 1987b), N-(ethoxycarbonyl)-2-ethoxy1,2-dihydroquinoline (EEDQ) (Phelps & Hatefi, 1984b), [ @-fluorosulfonyl)benzoyl]-5’-adenosine (FSBA) (Phelps & Hatefi, 1985; Wakabayashi & Hatefi, 1987a), ethoxyformic anhydride (Yamaguchi & Hatefi, 1985), dansyl chloride (Yamaguchi & Hatefi, 1985), tetranitromethane (Wu & Fisher, 1982), pyridoxal phosphate (Yamaguchi & Hatefi, 1985), butanedione and phenylglyoxal (Djavadi-Ohaniance & Hatefi, 1975), and 4-chloro-7-nitrobenzofurazan(Nbf-CI) (Persson et al., 1988). In addition, the enzyme activity is highly sensitive to trypsin (Juntti et al., 1970; Djavadi-Ohaniance & Hatefi, 1975; Blazyk et al., 1976). Substrates exhibit different effects on inhibition of transhydrogenase activity by these reagents, from strong protection against inhibition

0 1989 American Chemical Society

Nicotinamide Nucleotide Transhydrogenase (Phelps & Hatefi, 1984a, 1985; Yamaguchi & Hatefi, 1985) to considerable stimulation of the inhibition rate constant (Blazyk et al., 1976; Earle et al., 1978; Yamaguchi & Hatefi, 1985). The latter effect has been of particular interest, because it suggests substrate-induced enzyme conformation change, a feature that may be relevant to the mechanism of proton translocation by this energy-transducing enzyme. In this regard, the substrate of interest has been NADPH. It has been shown that its presence in the reaction mixture increases the inhibition rate of the enzyme by thiol modifiers, trypsin, FSBA, DCCD, ethoxyformic anhydride, dansyl chloride, and Nbf-C1 which appears to modify several sulfhydryl groups (Persson et al., 1988). The present study shows that NADP retards and NADPH accelerates the inhibition rate of purified bovine transhydrogenase by N-ethylmaleimide (NEM), that these effects on the NEM inhibition rate appear to be related to the opposite changes brought about by NADP and NADPH binding on the pK, of the NEM target concerned with enzyme inhibition, and that this target is the sulfhydryl group belonging to Cys-893, one of 1 1 cysteine residues of the enzyme (Yamaguchi et al., 1988a,b). Cys-893 is located 113 residues upstream of the tyrosine modified by FSBA, apparently at the NADP(H) binding site of the enzyme (Wakabayashi & Hatefi, 1987a). MATERIALS AND METHODS Materials. N-[Eth~L2-~H]maleimide (41.3 Ci/mmol) was obtained from NEN. Unlabeled N-ethylmaleimide, DL-dithiothreitol, Tris, methyl methanethiosulfonate (MMTS), NMN, NMNH, and 3-acetylpyridine adenine dinucleotide were obtained from Sigma. NAD, NADH, NADP, and NADPH were obtained from Calbiochem, and diethanolamine was from Baker. Bovine mitochondrial transhydrogenase was prepared as described by Phelps and Hatefi (1984). The specific activity of the enzyme used was 18-20 pmol of AcPyAD reduced by NADPH min-l (mg of protein)-' at 37 OC. Assay of Transhydrogenase Activity. Transhydrogenation from NADPH to AcPyAD was assayed at 37 "C in a reaction mixture containing 100 mM sodium phosphate, pH 6.5, and 0.33 mM each of NADPH and AcPyAD. The reaction was started by the addition of enzyme, and the reduction of AcPyAD was followed at 375 minus 425 nm in an Aminco DW2a dual-wavelength spectrophotometer. Rates were calculated by using a value of 6.38 mM-' cm-' for the absorbance difference of AcPyADH and NADPH at the above wavelength pair (Phelps & Hatefi, 1981). Protein concentration was determined by the method of Peterson (1977), using bovine serum albumin as standard. Modifcation of the Enzyme with N E M . The standard reaction mixture for NEM modification contained the enzyme (10-20 pg of protein) and 0.04 pmol of NEM in the absence or presence of 0.02 pmol of substrate in 100-pL final volume of 50 mM Tris-acetate, pH 7.5, containing 0.001% potassium cholate. The mixture was incubated at 23 OC, and the residual enzyme activity was measured by using aliquots of the reaction mixture at appropriate time intervals. The time course of the inactivation followed pseudo-first-order kinetics, and the inhibition rate constants ( k ) were calculated from the slopes of semilogarithmic plots, using the equation In ( V / V o )= -kt, where Vis the residual activity at time t and Vois the original activity. Modification of the Enzyme with [314NEM. The reaction mixture contained 0.76 mg of the enzyme, 0.4 mM [3H]NEM (- lo7 cpm/Mmol) in the absence or presence of 0.2 mM NADPH, and 50 mM Tris-acetate, pH 7.5, containing 0.001%

Biochemistry, Vol. 28, No. 14, 1989 6051 potassium cholate up to a final volume of 0.5 mL. The reaction mixture was incubated at 23 OC for 75 s, and the reaction was quenched by the addition of 10 pL of 0.4 M dithiothreitol. The residual enzymatic activity was measured by using an aliquot of the reaction mixture. The unbound [3H]NEM and other additives were removed by passing the reaction mixture through a Sephadex G-50 column (1.1 X 7 cm) equilibrated with the same buffer, and the modified enzyme was lyophilized. Cyanogen Bromide Fragmentation. The lyophilized protein was dissolved in 0.5 mL of 70% formic acid, containing 4 mg of cyanogen bromide, and the solution was incubated at room temperature for 20 h. Then, 5 mL of water was added and the solution lyophilized again. Separation of Peptides by Reverse-Phase Column Chromatography. For isolation of peptides, the Pharmacia FPLC system and the reverse-phase column ProRPC HR 5/10 (5 X 100 mm) were used. The cyanogen bromide treated and lyophilized material was dissolved in 0.7 mL of 0.1% trifluoroacetic acid and applied to a reverse-phase column equilibrated with the same buffer. Peptides were eluted by a linear gradient of a 2-propanol/acetonitrile (1 :2) mixture containing 0.1% trifluoroacetic acid, and the elution of peptides was monitored at 214 nm. The flow rate was 0.5 mL/min, and fractions of 0.5 mL each were collected. The fractions with high radioactivity were collected, lyophilized, dissolved in 0.7 mL of 0.1% trifluoroacetic acid, and applied to the same reverse-phase column equilibrated with 0.1% trifluoroacetic acid. Peptides were eluted by a linear gradient of a 2propanol/acetonitrile (2: 1) mixture containing 0.1% trifluoroacetic acid. Each peak fraction was collected, its radioactivity was measured, and the radioactive peak fractions were lyophilized. If the peptide isolated was not pure at this stage, the lyophilized sample was loaded onto the reverse-phase column again, and peptides were eluted once more by a linear gradient of a 2-propanol/acetonitrile (1 :2) mixture containing 0.1% trifluoroacetic acid. The major radioactive peak was collected, lyophilized, and subjected to amino acid and Nterminal sequence analysis. Amino Acid and Sequence Analysis of Peptides. Manual Edman degradation was carried out using (dimethylamino) azobenzene isothiocyanate. The (dimethy1amino)azobenzene thiohydantoin derivatives were identified by two-dimensional thin-layer chromatography (Chan, 1983). For amino acid analysis, the peptide in 6 N HCl was sealed in an ampule under vacuum and hydrolyzed at 110 OC for 72 h. Amino acid analysis was performed in a Beckman Model 121-M amino acid analyzer. Modification of Transhydrogenase with MMTS. The enzyme (55 pg of protein) was incubated for 2 min at 23 OC with 0.25 mM MMTS in 40 p L of 50 mM Tris-acetate, pH 7.5, containing 0.001% potassium cholate. This treatment decreased the specific activity of the enzyme from 18 to 4.3 pmol of AcPyAD reduced min-' (mg of protein)-'. Longer incubations and higher MMTS concentrations did not result in greater inactivation. RESULTS Inhibition of Purijied Transhydrogenase by N E M and Effect of Substrates. Figure 1 shows in semilogarithmic plots the inhibition time course of purified transhydrogenase when incubated with several concentrations of NEM. In the range of NEM concentration used, the inactivation time course followed pseudo-first-order kinetics, and the reaction order with respect to [NEM] was unity (see the slope of the line in the inset of Figure I ) , suggesting that inhibition results from

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Biochemistry, Vol. 28, No. 14, 1989

Yamaguchi and Hatefi

Incubation Time (min) 1

2

3

4

5 1.2

-k.-

0.8

E Y

0.4

NADPH ImMP I

1

I

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I

0.1

0.2

0.3

0.4

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NADPH (mM) Effect of NADPH concentration on the rate constant of transhydrogenase inactivation by NEM. The enzyme (20 pg of protein) was incubated at 23 OC with 0.4 mM NEM and the NADPH concentrations shown in 100-pLfinal volume of 50 mM Tris-acetate, pH 7.5, containing 0.001% potassium cholate. At proper intervals, 10-pL aliquots were withdrawn and assayed for residual enzyme activity, and the NEM inhibition rate constant ( k ) was determined at each NADPH concentration. The inset shows a double-reciprocal plot of kb - k, versus NADPH concentration, where kb is the rate constant in the presence of varying concentrations of NADPH and k, is the rate constant in the absence of NADPH. FIGURE 2:

-Log [NEM] Inhibition time course of transhydrogenase activity in the presence of NEM. The enzyme (IO pg of protein) was incubated at 23 O C in 100-pL final volume of 50 mM Tris-acetate buffer, pH 7.5, containing 0.001% potassium cholate, with 0 (0),0.06 (O), 0.1 ( O ) , 0.2 (M),0.4 (A),0.6 (A),and 1.0 (v)mM NEM for the indicated times and assayed for the residual activity as described under Materials and Methods. Voand Vare reaction rates at time zero and the times shown. ko and k are pseudo-first-order rate constants, respectively, in the absence and presence of the inhibitor concentrations indicated. The inset shows a plot of the logarithm of ( k - b)versus the logarithm of the corresponding NEM concentrations. The slope of the line (n) shows the reaction order with respect to inhibitor concentration. FIGURE 1 :

Table I: Effects of Substrates on the Rate of Inactivation of Purified Nicotinamide Nucleotide Transhydrogenase by NEMO additions k - k, (min-l) additions k - k , (min-l) 0.253 2’-AMP 0.150 none NAD 0.253 NMN 0.230 NADH 0.200 NMNH 0.253 NADP 0.115 2’-AMP + NMN 0.152 NADPH 1.800 2’-AMP + NMNH 0.168 5‘-AMP 0.258 “The enzyme (10 pg of protein in 100 p L of 50 mM Tris-acetate, pH 7.5, containing 0.001% potassium cholate) was incubated with 0.4 mM NEM in the absence or the presence of 0.2 mM concentration of each substrate or analogue. The rate constant ( k - k,) for NEM inactivation of the enzyme was determined as described in the legend to Figure I .

inter Action of one molecule of NEM with one active unit of the enzyme (Levy et al., 1963). Table I shows the effects of substrates on inactivation of purified transhydrogenase by NEM. At neutral pH, NADP partially protected the enzyme against NEM inactivation, while NADPH greatly accelerated the rate of inactivation. Both of these effects were highly pH dependent, however, as will be seen below. NAD had no effect, and NADH showed a slight protective effect. 2’-AMP, an

analogue of NADP, exhibited a protective effect similar to that shown by NADP, whereas 5‘-AMP, an analogue of NAD, showed no effect. Neither N M N nor N M N H had any effect, and the effect of 2’-AMP plus N M N or N M N H was no more than that shown by 2’-AMP alone. Since NADPH accelerated the NEM inactivation rate by severalfold, the effect of NADPH concentration on the NEM inactivation rate was examined. Figure 2 shows the results. The stimulating effect of NADPH was saturable, and double-reciprocal analysis of the data (Figure 2, inset) indicated a half-saturation concentration for NADPH of 13.4 pM. This value is essentially the same as the reported Michaelis constant (20 pM) of the enzyme for NADPH at pH 7.5 (Teixeira da Cruz et al., 1971). The reaction order with respect to [NEM] in the presence of 0.2 mM NADPH was estimated also to be unity (see Figure l ) , which suggested that the greatly facilitated inhibition in the presence of NADPH still involved modification by NEM of a single residue (possibly the same thiol as in the absence of NADPH) per active unit of the transhydrogenase. Effect of p H on NEM Inhibition. The rate of enzyme inhibition by NEM was highly pH dependent, both in the absence and in the presence of NADP(H) in the incubation mixture. As seen in Figure 3, the inhibition rate constant was small at pH