Mitochondrial nicotinamide nucleotide transhydrogenase: NADPH binding increases and NADP binding decreases the acidity and susceptibility to modification of cysteine-893

The mitochondrial nicotinamide nucleotide transhydrogenase is a dimeric enzyme of monomer Mr 110,000. 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. Int. 15, 915-924]. N-Ethylmaleimide, as well as other sulfhydryl group modifiers, inhibits the transhydrogenase. The presence of NADP in the incubation mixture suppressed the inhibition rate by N-ethylmaleimide, and the presence of NADPH greatly increased it. NAD and NADH had little or no effect. The NADPH effect was concentration dependent and saturable, with a half-maximal NADPH concentration effect close to the Km of the enzyme for NADPH. Study of the effect of pH on the N-ethylmaleimide inhibition rate showed that NADPH binding by the enzyme lowers the apparent pKa of the N-ethylmaleimide-sensitive group by 0.4 of a pH unit and NADP binding raises this pKa by 0.4 of a 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 113 residues upstream of the tyrosyl residue modified by [p-(fluorosulfonyl)benzoyl]-5'-adenosine at the putative NADP(H) binding site of the enzyme (see above references).(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational features of bovine heart mitochondrial transhydrogenase

Both purified and functionally reconstituted bovine heart mitochondrial transhydrogenase were treated with various sulfhydryl modification reagents in the presence of substrates. In all cases, NAD+ and NADH had no effect on the rate of inactivation. NADP+ protected transhydrogenase from inactivation by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in both systems, while NADPH slightly protected the reconstituted enzyme but stimulated inactivation in the purified enzyme. The rate of N-ethylmaleimide (NEM) inactivation was enhanced by NADPH in both systems. The copper-(o-phenanthroline)2 complex [Cu(OP)2] inhibited the purified enzyme, and this inhibition was substantially prevented by NADP+. Transhydrogenase was shown to undergo conformational changes upon binding of NADP+ or NADPH. Sulfhydryl quantitation with DTNB indicated the presence of two sulfhydryl groups exposed to the external medium in the native conformation of the soluble purified enzyme or after reconstitution into phosphatidylcholine liposomes. In the presence of NADP+, one sulfhydryl group was quantitated in the nondenatured soluble enzyme, while none was found in the reconstituted enzyme, suggesting that the reactive sulfhydryl groups were less accessible in the NADP+-enzyme complex. In the presence of NADPH, however, four sulfhydryl groups were found to be exposed to DTNB in both the soluble and reconstituted enzymes. NEM selectively reacted with only one sulfhydryl group of the purified enzyme in the absence of substrates, but the presence of NADPH stimulated the NEM-dependent inactivation of the enzyme and resulted in the modification of three additional sulfhydryl groups. The sulfhydryl group not modified by NEM in the absence of substrates is not sterically hindered in the native enzyme as it can still be quantitated by DTNB or modified by iodoacetamide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional cysteinyl residues in human placental aldose reductase

Incubation of human placental aldose reductase (EC 1.1.1.21) with the sulfhydryl oxidizing reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM) results in a biexponential loss of catalytic activity. Inactivation by DTNB or NEM is prevented by saturating concentrations of NADPH. ATP-ribose offers partial protection against inactivation by DTNB, whereas NADP, nicotinamide mononucleotide (NMN), and the substrates glyceraldehyde and glucose offer little or no protection. The inactivation by DTNB was reversed by dithiothreitol and partially by 2-mercaptoethanol but not by KCN. When the release of 2-nitro-5-mercaptobenzoic acid was measured, 3 mol of sulfhydryl residues was found to be modified per mole of the enzyme by DTNB. Correlation of the fractional activity remaining with the extent of modification by the statistical method of C.-L. Tsou (1962, Sci. Sin. 11, 1535-1558) indicates that of the three reactive residues, one reacts at a faster rate than the other two, and that two residues are essential for the catalytic activity of the enzyme. Labeling of the total sulfhydryl by [14C]NEM and quantification of DTNB-reactive residues in the enzyme denatured by 6 M urea indicates that a total of seven sulfhydryl residues are present in the protein. The modification of the enzyme did not affect Km glyceraldehyde, but the modified enzyme had a lower Km NADPH. Kinetic analysis of the data suggests that a biexponential nature of inactivation could be due to the formation of a dissociable E:DTNB complex and the presence of a partially active enzyme species.     

Thermal inactivation of reduced ferredoxin (flavodoxin):NADP+ oxidoreductase from Escherichia coli

Ferredoxin (flavodoxin):NADP+ oxidoreductase (FNR) is an essential enzyme that supplies electrons from NADPH to support flavodoxin-dependent enzyme radical generation and enzyme activation. FNR is a monomeric enzyme that contains a non-covalently bound FAD cofactor. We report that reduced FNR from Escherichia coli is subject to inactivation due to unfolding of the protein and dissociation of the FADH(2) cofactor at 37 degrees C. The inactivation rate is temperature-dependent in a manner that parallels the thermal unfolding of the protein and is slowed by binding of ferredoxin or flavodoxin. Understanding factors that minimize inactivation is critical for utilizing FNR as an accessory protein for S-adenosyl-L-methionine-dependent radical enzymes and manipulating FNR as an electron source for biotechnology applications.     

Characterization of an active site peptide modified by the substrate analogue 3-bromo-2-ketoglutarate on a single chain of dimeric NADP+-dependent isocitrate dehydrogenase

The substrate analogue 3-bromo-2-ketoglutarate reacts with pig heart NADP+-dependent isocitrate dehydrogenase to yield partially inactive enzyme. Following 65% inactivation, no further inactivation was observed. Concomitant with this inactivation, incorporation of 1 mol of reagent/mol of enzyme dimer was measured. The dependence of the inactivation rate on bromoketoglutarate concentration is consistent with reversible binding of reagent (KI = 360 microM) prior to irreversible reaction. Manganous isocitrate reduces the rate of inactivation by 80% but does not provide complete protection even at saturating concentrations. Complete protection is obtained with NADP+ or the NADP+-alpha-ketoglutarate adduct. By modification with [14C]bromoketoglutarate or by NaB3H4 reduction of modified enzyme, a single major radiolabeled tryptic peptide was obtained by high performance liquid chromatography with the sequence: Asp-Leu-Ala-Gly-X-Ile-His-Gly-Leu-Ser-Asn-Val-Lys. Evidence in the following paper (Bailey, J.M., Colman, R.F. (1987) J. Biol. Chem. 262, 12620-12626) indicates that X is glutamic acid. Enzyme modified at the coenzyme site by 2-(bromo-2,3-dioxobutylthio)-1,N(6)-ethenoadenosine 2',5'-biphosphate in the presence of manganous isocitrate is not further inactivated by bromoketoglutarate. Bromoketoglutarate-modified enzyme exhibits a stoichiometry of binding isocitrate and NADPH equal to 1 mol/mol of enzyme dimer, half that of native enzyme. These results indicate that bromoketoglutarate modifies a residue in the nicotinamide region of the coenzyme site proximal to the substrate site and that reaction at one catalytic site of the enzyme dimer decreases the activity of the other site.     

Electron transfer by ferredoxin:NADP+ reductase. Rapid-reaction evidence for participation of a ternary complex

Rapid reaction studies presented herein show that ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2) catalyzes electron transfer from spinach ferredoxin (Fd) to NADP+ via a ternary complex, Fd X FNR X NADP+. In the absence of NADP+, reduction of ferredoxin:NADP+ reductase by Fd was much slower than the catalytic rate: 37-80 s-1 versus at least 445 e-s-1; dissociation of oxidized spinach ferredoxin (Fdox) from one-electron reduced ferredoxin:NADP+ reductase (FNRsq) limited the reduction of FNR. This confirms the steady-state kinetic analysis of Masaki et al. (Masaki, R., Yoshikaya, S., and Matsubara, H. (1982) Biochim. Biophys. Acta 700, 101-109). Occupation of the NADP+ binding site of FNR by NADP+ or by 2',5'-ADP (a nonreducible NADP+ analogue) greatly increased the rate of electron transfer from Fd to FNR, releiving inhibition by Fdox. NADP+ (and 2',5'-ADP) probably facilitate the dissociation of Fdox; equilibrium studies have shown that nucleotide binding decreases the association of Fd with FNR (Batie, C. J. (1983) Ph.D. dissertation, Duke University; Batie, C. J., and Kamin, H. (1982) in Flavins and Flavoproteins VII (Massey, V., and Williams, C. H., Jr., eds) pp. 679-683, Elsevier, New York; Batie, C.J., and Kamin, H. (1982) Fed. Proc. 41, 888; and Batie, C.J., and Kamin, H. (1984) J. Biol. Chem. 259, 8832-8839). Premixing Fd with FNR was found to inhibit the reaction of the flavoprotein with NADP+ and with NADPH; thus, substrate binding may be ordered, NADP+ first, then Fd. FNRred and NADP+ very rapidly formed an FNRred X NADP+ complex with flavin to nicotinamide charge transfer bands. The Fdred X NADP+ complex then relaxed to an equilibrium species; the spectrum indicated a predominance of FNRox X NADPH charge-transfer complex. However, charge-transfer species were not observed during turnover; thus, their participation in catalysis of electron transfer from Fd to NADP+ remains uncertain. The catalytic rate of Fd to NADP+ electron transfer, as well as the rates of electron transfer from Fd to FNR, and from FNR to NADP+ were decreased when the reactants were in D2O; diaphorase activity was unaffected by solvent. On the basis of the data presented, a scheme for the catalytic mechanism of catalysis by FNR is presented.     

Structural backgrounds for the formation of a catalytically competent complex with NADP(H) during hydride transfer in ferredoxin-NADP(+) reductases

The role of the highly conserved C266 and L268 of pea ferredoxin-NADP(+) reductase (FNR) in formation of the catalytically competent complex of the enzyme with NADP(H) was investigated. Previous studies suggest that the volume of these side-chains, situated facing the side of the C-terminal Y308 catalytic residue not stacking the flavin isoalloxazine ring, may be directly involved in the fine-tuning of the catalytic efficiency of the enzyme. Wild-type pea FNR as well as single and double mutants of C266 and L268 residues were analysed by fast transient-kinetic techniques and their midpoint reduction potentials were determined. For the C266A, C266M and C266A/L268A mutants a significant reduction in the overall hydride transfer (HT) rates was observed along with the absence of charge-transfer complex formation. The HT rate constants for NADPH oxidation were lower than those for NADP(+) reduction, reaching a 30-fold decrease in the double mutant. In agreement, these variants exhibited more negative midpoint potentials with respect to the wild-type enzyme. The three-dimensional structures of C266M and L268V variants were solved. The C266M mutant shows a displacement of E306 away from the relevant residue S90 to accommodate the bulky methionine introduced. The overall findings indicate that in FNR the volume of the residue at position 266 is essential to attain the catalytic architecture between the nicotinamide and isoalloxazine rings at the active site and, therefore, for an efficient HT process. In addition, flexibility of the 268-270 loop appears to be critical for FNR to achieve catalytically competent complexes with NADP(H).     

L-cystine inhibits aspartate-beta-semialdehyde dehydrogenase by covalently binding to the essential 135Cys of the enzyme

Aspartate-beta-semialdehyde dehydrogenase (ASADH) from Escherichia coli is inhibited by L- and D-cystine, and by other cystine derivatives. Enzyme inhibition is quantitatively reversed by addition of dithiothreitol (DTT), dithioerythrytol, beta-mercaptoethanol, di-mercaptopropanol or glutathione to the cystine-inactivated enzyme. Cystine labeling of the enzyme is a pH dependent process and is optimal at pH values ranging from 7.0 to 7.5. Both the cysteine incorporation profile and the inactivation curve of the enzyme as a function of pH suggest that a group(s) with pK(a) of 8.5 could be involved in cystine binding. Stoichiometry of the inactivation reaction indicates that one cysteine residue from the enzyme subunit is reactive against cystine, as found by direct incorporation of radioactive cystine into the enzyme and by free-thiol titration of the enzyme with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) before and after the cystine treatment. One mole of cysteine is released from each mol of cystine after reaction with the enzyme. ASA, NADP and NADPH did not prevent cystine inhibition. The [35S]cysteine-labelled enzyme can be visualized after electrophoresis in polyacrylamide gels and further detection by autoradiography. After pepsin treatment of the [35S]cysteine-inactivated enzyme, a main radioactive peptide was isolated by HPLC. The amino acid sequence of this peptide was determined as FVGGN(Cys)(2)TVSL, thus demonstrating that the essential 135Cys is the amino acid residue modified by the treatment with cystine.     

Evidence for the Existence of Two Essential and Proximal Cysteinyl Residues in NADP-Malic Enzyme from Maize Leaves

Incubation of maize (Zea mays) leaf NADP-malic enzyme with monofunctional and bifunctional N-substituted maleimides results in an irreversible inactivation of the enzyme. Inactivation by the monofunctional reagents, N-ethylmaleimide (NEM) and N-phenylmaleimide, followed pseudo-first-order kinetics. The maximum inactivation rate constant for phenylmaleimide was 10-fold higher than that for NEM, suggesting a possible hydrophobic microenvironment of the residue(s) involved in the modification of the enzyme. In contrast, the inactivation kinetics with the bifunctional maleimides, ortho-, meta-, and para-phenylenebismaleimide, were biphasic, probably due to different reactivities of the groups reacting with the two heads of these bifunctional reagents, with a possible cross-linking of two sulfhydryl groups. The inactivation by mono and bifunctional maleimides was partially prevented by Mg²⁺ and l-malate, and NADP prevented the inactivation almost totally. Determination of the number of reactive sulfhydryl groups of the native enzyme with [³H]NEM in the absence or presence of NADP showed that inactivation occurred concomitantly with the modification of two cysteinyl residues per enzyme monomer. The presence of these two essential residues was confirmed by titration of sulfhydryl groups with [³H]NEM in the enzyme previously modified by o-phenylenebismaleimide in the absence or presence of NADP.     

A lysyl residue at the NADP binding site of ferredoxin-NADP reductase.

Dansyl chloride, at low molar ratio, inactivates ferredoxin-NADP reductase (NADPH:ferredoxin oxidoreductase, EC 1.6.7.1). The complete protection afforded either by NADP or NADPH suggests a direct involvement of the active site. Experiments with [Me-14C] dansyl chloride showed that about 1.5 residues per flavin were dansylated: by differential labelling experiments using NADP, it has been proved that enzyme inactivation is due to dansylation of one residue. The group modified has been identified as the epsilon-amino group of a lysine. The pH-inactivation profile indicates that this essential group has an apparent pKa of 8.7. The dansylated flavoprotein seems to maintain its native conformation; it shows a fluorescent chromophore with a peak at 335 nm. The modified enzyme has lost the capacity to form a complex with NADP, nevertheless it interacts normally with ferredoxin. It is concluded that the loss of catalytic activity which parallels the dansylation of a lysyl residue occurs because this residue is essential for the binding of the pyridine nucleotide substrate. Protection experiments with a series of coenzyme analogs further indicate that this lysyl residue interacts, most likely, with the 2'-phosphate moiety of NADP(H).     

l-Cystine inhibits aspartate-β-semialdehyde dehydrogenase by covalently binding to the essential 135Cys of the enzyme

Aspartate-β-semialdehyde dehydrogenase (ASADH) from Escherichia coli is inhibited by l- and d-cystine, and by other cystine derivatives. Enzyme inhibition is quantitatively reversed by addition of dithiothreitol (DTT), dithioerythrytol, β-mercaptoethanol, di-mercaptopropanol or glutathione to the cystine-inactivated enzyme. Cystine labeling of the enzyme is a pH dependent process and is optimal at pH values ranging from 7.0 to 7.5. Both the cysteine incorporation profile and the inactivation curve of the enzyme as a function of pH suggest that a group(s) with pK_a of 8.5 could be involved in cystine binding. Stoichiometry of the inactivation reaction indicates that one cysteine residue from the enzyme subunit is reactive against cystine, as found by direct incorporation of radioactive cystine into the enzyme and by free-thiol titration of the enzyme with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) before and after the cystine treatment. One mole of cysteine is released from each mol of cystine after reaction with the enzyme. ASA, NADP and NADPH did not prevent cystine inhibition. The [³⁵S]cysteine-labelled enzyme can be visualized after electrophoresis in polyacrylamide gels and further detection by autoradiography. After pepsin treatment of the [³⁵S]cysteine-inactivated enzyme, a main radioactive peptide was isolated by HPLC. The amino acid sequence of this peptide was determined as FVGGN(Cys)₂TVSL, thus demonstrating that the essential ¹³⁵Cys is the amino acid residue modified by the treatment with cystine.     

The reaction of N-ethylmaleimide at the active site of succinate dehydrogenase.

Since 1938 mammalian succinate dehydrogenase has been thought to contain thiol groups at the active site. This hypothesis was questioned recently, because irreversible inhibition by bromopyruvate and N-ethylmaleimide appeared not to satisfy the requisite criteria for reaction at the active site. These recent observations of incomplete inactivation of succinate dehydrogenase by N-ethylmaleimide and incomplete protection by substrates can, however, be explained adequately by the presence of oxalacetate and other strong competitors of the inactivation process in the enzyme used in these studies. Substrates, competitive inhibitors, and anions which activate succinate dehydrogenase protect the enzyme from inhibition by N-ethylmaleimide. Inhibition of succinate dehydrogenase by N-ethylmaleimide involves at least two second order reactions which are pH dependent, with pKa values of 8.0 to 8.2. This pH dependence, the known reactivity of N-ethylmaleimide toward thiols, and the protection by substrate and competitive inhibitors indicate that sulfhydryl residues are required for catalytic activity and perform an essential, not secondary, role in the catalysis. Just as the presence of tightly bound oxalacetate prevents inhibition by N-ethylmaleimide, alkylation of the sulfhydryl residue(s) at the active site prevents the binding of [14C]oxalacetate. Thus, these thiol groups at the active site also may be the site of tight binding of oxalacetate during the activation-deactivation cycle.     

Presence of essential histidine residues in nadp-malic enzyme from maize

Modification of maize leaf NADP-malic enzyme by diethylpyrocarbonate (DEP) caused rapid and complete inactivation of the enzyme. The inactivation followed pseudo-first-order reaction kinetics. The inactivation of the enzyme showed saturation kinetics with a half inactivation time, at saturating DEP, equal to 0.15 min and K_DEP = 20 mM. The rate of inactivation was faster at 25° as compared to 0° (t_0.5 0.75 min at 25° as against 5.6 min at 4° at 5 mM DEP). The enzyme was partially protected against DEP inactivation by NADP and complete protection was seen in the presence of NADP + Mg²⁺ + malate or its analogues, thereby indicating that DEP modifies the active site. The modified enzyme showed an increase in absorbance at 240 nm which was lost upon treatment with 0.25 M NH₂OH and almost complete recovery of the enzyme activity was also observed. The results suggest that DEP modifies 3.0 residues per subunit and of these at least two residue per subunit can be modified without loss of activity in the presence of substrate. Modification of about one histidine residue is correlated with the loss of enzyme activity.     

Involvement of thiol groups in the activity of phosphoenolpyruvate carboxylase from maize leaves

Purified maize leaf phosphoenolpyruvate carboxylase (EC 4.1.1.31) was completely inactivated by several thiol-modifying reagents, including, CuCl₂, CdCl₂ and N-ethylmaleimide. The inactivation by CuCl₂ could be reversed by dithiothreitol, suggesting the involvement of vicinal dithiols in the inactivation process.Complete inactivation of phosphoenolpyruvate carboxylase was correlated with the incorporation of two mol (³H)N-ethylmaleimide per 100-kilodalton subunit. The total protection of the enzyme against N-ethylmaleimide inactivation afforded by the substrate, phosphoenolpyruvate, was correlated with the protection of one mol (³H)N-ethylmaleimide reactive residue per mol subunit.The complete inactivation of phosphoenolpyruvate carboxylase by N-ethylmaleimide and the protection afforded by phosphoenolpyruvate against modification suggest the presence of an essential cysteine residue in the catalytic site of the C₄ leaf enzyme.Abbreviations PEP, phosphoenolpyruvate - Mops, 4-morpholinepropanesulphonic acid (Consejo Nacional de Investigaciones Científicas y Técnicas, Fundación M. Lillo y U.N. de Rosario).     

Essential histidyl residues of ferredoxin-NADP+ oxidoreductase revealed by diethyl pyrocarbonate inactivation

Diethyl pyrocarbonate inhibited diaphorase activity of ferredoxin-NADP+ oxidoreductase with a second-order rate constant of 2 mM-1 X min-1 at pH 7.0 and 20 degrees C, showing a concomitant increase in absorbance at 242 nm due to formation of carbethoxyhistidyl derivatives. Activity could be restored by hydroxylamine, and the pH curve of inactivation indicated the involvement of a residue having a pKa of 6.8. Derivatization of tyrosyl residues was also evident, although with no effect on the diaphorase activity. Both NADP+ and NADPH protected the enzyme against inactivation, suggesting that the modification occurred at or near the nucleotide binding domain. The reductase lost all of its diaphorase activity after about two histidine residues had been blocked by the reagent. In differential-labeling experiments with NADP+ as protective agent, it was shown that diaphorase inactivation resulted from blocking of only one histidyl residue per mole of enzyme. Modified reductase did not bind pyridine nucleotides. Modification of the flavoprotein in the presence of NADP+, i.e., with full preservation of diaphorase activity, resulted in a significant impairment of cytochrome c reductase activity, with a second-order rate constant for inactivation of about 0.5 mM-1 X min-1. Reversal by hydroxylamine and spectroscopic data indicated that this second residue was also a histidine. Ferredoxin afforded only slight protection against this inhibition. Conversely, carbethoxylation of the enzyme did not affect complex formation with the ferrosulfoprotein. Redox titration of the modified reductase with NADPH and with reduced ferredoxin suggested that the second histidine might be located in the electron pathway between FAD and ferredoxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mitochondrial nicotinamide nucleotide transhydrogenase: active site modification by 5'-[p-(fluorosulfonyl)benzoyl]adenosine

Membrane-bound and purified mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) was inhibited by incubation with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA), which is an analogue of TH substrates and their competitive inhibitors, namely, 5'-, 2'-, or 3'-AMP. NAD(H) and analogues, NADP, 5'-AMP, 5'-ADP, and 2'-AMP/3'-AMP mixed isomers protected TH against inhibition by FSBA, but NADPH accelerated the inhibition rate. In the absence of protective ligands or in the presence of NADP, FSBA appeared to modify the NAD(H) binding site of TH, because, unlike unmodified TH, the enzyme modified by FSBA under these conditions did not bind to an NAD-affinity column (NAD-agarose). However, when the NAD(H) binding site of TH was protected in the presence of 5'-AMP or NAD, then FSBA modification resulted in an inhibited enzyme that did bind to NAD-agarose, suggesting FSBA modification of the NADP(H) binding site or an essential residue outside the active site. [3H]FSBA was covalently bound to TH, and complete inhibition corresponded to the binding of about 0.5 mol of [3H]FSBA/mol of TH. Since purified TH is known to be dimeric in the isolated state, this binding stoichiometry suggests half-of-the-sites reactivity. A similar binding stoichiometry was found earlier for complete inhibition of TH by [14C]DCCD [Phelps, D.C., & Hatefi, Y. (1984) Biochemistry 23, 4475-4480]. The active site directed labeling of TH by radioactive FSBA should allow isolation of appropriate peptides for sequence analysis of the NAD(H) and possibly the NADP(H) binding domains.     

Oxalacetate decarboxylase and pyruvate carboxylase activities, and effect of sulfhydryl reagents in malic enzyme from Sulfolobus solfataricus

Malic enzyme (S)-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40) purified from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4, catalyzed the metal-dependent decarboxylation of oxaloacetate at optimum pH 7.6 at a rate comparable to the decarboxylation of L-malate. The oxaloacetate decarboxylase activity was stimulated about 50% by NADP but only in the presence of MgCl2, and was strongly inhibited by L-malate and NADPH which abolished the NADP activation. In the presence of MnCl2 and in the absence of NADP, the Michaelis constant and Vm for oxaloacetate were 1.7 mM and 2.3 mumol.min-1.mg-1, respectively. When MgCl2 replaced MnCl2, the kinetic parameters for oxaloacetate remained substantially unvaried, whereas the Km and Vm values for L-malate have been found to vary depending on the metal ion. The enzyme carried out the reverse reaction (malate synthesis) at about 70% of the forward reaction, at pH 7.2 and in the presence of relatively high concentrations of bicarbonate and pyruvate. Sulfhydryl residues (three cysteine residues per subunit) have been shown to be essential for the enzymatic activity of the Sulfolobus solfataricus malic enzyme. 5,5'-Dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate and N-ethylmaleimide caused the inactivation of the oxidative decarboxylase activity, but at different rates. The inactivation of the overall activity by p-hydroxymercuribenzoate was partially prevented by NADP singly or in combination with both L-malate and MnCl2, and strongly enhanced by the carboxylic acid substrates; NADP + malate + MnCl2 afforded total protection. The inactivation of the oxaloacetate decarboxylase activity by p-hydroxymercuribenzoate treatment was found to occur at a slower rate than that of the oxidative decarboxylase activity.     

The Kinetics of N-Ethylmaleimide Inhibition of a Vacuolar H+-ATPase and Determination of Nucleotide Dissociation Constants

All eukaryotic vacuolar (V-type) ATPases share the property of being inhibited by low concentrations (1-2 [mu]M) if N-ethylmaleimide (NEM). This distinguishes them from P-type ATPases, which are inhibited by higher concentrations of NEM (0.1-1 mM), and F-type ATPases, which are virtually resistant to inhibition by NEM. Using tonoplast vesicles from Beta vulgaris we have determined the kinetics of NEM inactivation of the V-type ATPase to be pseudo-first order. The concentration dependence of the reaction indicates interaction with a single class of inhibitory site with a rate constant of 4.1 x 104 M-1 min-1. Nucleotides protect against inactivation with an efficacy that agrees with their capacity to act as enzyme substrates. The dissociation constant for MgATP has been determined from protection experiments to be 0.44 mM, which is close to the observed Km for hydrolysis (0.39 mM). Likewise, the dissociation constant for protection by MgADP (127 [mu]M) is close to its inhibition constant as a competitive inhibitor (110 [mu]M). Taken together, these findings suggest that NEM inactivation is associated with nucleotide protectable exposure of a single cysteine residue on the catalytic subunit and confirm the utility of this residue for the determination of ligand dissociation constants through protection of maleimide inhibition.     

Structural backgrounds for the formation of a catalytically competent complex with NADP(H) during hydride transfer in ferredoxin–NADP+ reductases

The role of the highly conserved C266 and L268 of pea ferredoxin–NADP⁺ reductase (FNR) in formation of the catalytically competent complex of the enzyme with NADP(H) was investigated. Previous studies suggest that the volume of these side-chains, situated facing the side of the C-terminal Y308 catalytic residue not stacking the flavin isoalloxazine ring, may be directly involved in the fine-tuning of the catalytic efficiency of the enzyme. Wild-type pea FNR as well as single and double mutants of C266 and L268 residues were analysed by fast transient-kinetic techniques and their midpoint reduction potentials were determined. For the C266A, C266M and C266A/L268A mutants a significant reduction in the overall hydride transfer (HT) rates was observed along with the absence of charge-transfer complex formation. The HT rate constants for NADPH oxidation were lower than those for NADP⁺ reduction, reaching a 30-fold decrease in the double mutant. In agreement, these variants exhibited more negative midpoint potentials with respect to the wild-type enzyme. The three-dimensional structures of C266M and L268V variants were solved. The C266M mutant shows a displacement of E306 away from the relevant residue S90 to accommodate the bulky methionine introduced. The overall findings indicate that in FNR the volume of the residue at position 266 is essential to attain the catalytic architecture between the nicotinamide and isoalloxazine rings at the active site and, therefore, for an efficient HT process. In addition, flexibility of the 268–270 loop appears to be critical for FNR to achieve catalytically competent complexes with NADP(H).