Chemical modification of histidine residues in rabbit liver glutathione reductase

The role of histidine residues of glutathione reductase from rabbit liver was investigated by chemical modification with both ethoxyformic anhydride and dansyl chloride. At least four histidine residues were concomitantly modified by ethoxyformic anhydride at pH 6; both the GSSG reductase and the transhydrogenase activities were inhibited to the same extent. Dansyl chloride inactivated the enzyme showing pH-independence in the range 7-9. About 2.6 moles dansyl were incorporated in the protein 80% inactivated at pH 8, whereas at pH 7 a lower amount of labelling was found. Nearly complete reactivation of the inactivated enzyme could be obtained by incubation with hydroxylamine, which released all the acid-labile bound dansyl. Of the two histidine residues modified, only the slower reacting residue seems essential for activity. The modification with dansyl chloride will allow the identification of the histidine residues modified, in the sequence of the protein.     

The presence of a histidine residue at or near the NADPH binding site of enoyl reductase domain on the multifunctional fatty acid synthetase of chicken liver

Chemical modification of chicken liver fatty acid synthetase with the reagent ethoxyformic anhydride causes inactivation of the palmitate synthetase and enoyl reductase activities of the enzyme complex, but without significant effect on its beta-ketoacyl reductase or beta-ketoacyl dehydratase activity. The second-order rate constant of 0.2 mM-1 X s-1 for loss of synthetase activity is equal to the value for enoyl reductase, indicating that ethoxyformylation destroys the ability of the enzyme to reduce the unsaturated acyl intermediate. The specificity of this reagent for histidine residues is indicated by the appearance of a 240 nm absorption band for ethoxyformic histidine corresponding to the modification of 2.1 residues per enzyme dimer, and by the observation that the modified enzyme is readily reactivated by hydroxylamine. A pK value of 7.1 obtained by studies of the pH rate-profile of inactivation is consistent with that of histidine. Moreover, inactivation by ethoxyformic anhydride is unaffected by reversely blocking essential SH groups of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid), and therefore does not involve the reaction of these groups. The reaction of tyrosyl groups is excluded by an unchanged absorption at 278 nm. In other experiments, it was shown that inactivation of synthetase is protected by pyridine nucleotide cofactors and nucleotide analogs containing a 2'-phosphate group, and is accompanied by the loss of 2.4 NADPH binding sites. These results implicate the presence of a histidine residue at or near the binding site for 2'-phosphate group of pyridine nucleotide in the enoyl reductase domain of the synthetase.     

Ethoxyformylation of ribonuclease U2 form Ustilago sphaerogena.

RNase U2 was inactivated by incubation with ethoxyformic anhydride at pH 6.0 and pH 4.5. The absorbance of the RNase U2 increased at around 250 nm and decreased at around 280 nm. The inactivation occurred in parallel with the amount of modified histidine and plots of the relationship between the remaining activity and the modified histidine suggested that the modification of one of the two histidine residues totally inactivated the enzyme. The inactivated enzyme RNase U2 was reactivated by a low concentration of hydroxyamine, with removal of the ethoxyformyl group from the modified histidine residue. At pH 4.5, 2'-adenylate and 2'-guanylate protected RNase U2 from inactivation by ethoxyformic anhydride. The difference CD spectra showed that the ability of RNase U2 to form a complex with 2'-adenylate was lost on ethoxyformylation.     

Reversible inactivation of Saccharomyces cerevisiae glutathione reductase under reducing conditions

Glutathione reductase from Saccharomyces cerevisiae was rapidly inactivated following aerobic incubation with NADPH, NADH, and several other reductants, in a time- and temperature-dependent process. The inactivation had already reached 50% when the NADPH concentration reached that of the glutathione reductase subunit. The inactivation was very marked at pH values below 5.5 and over 7, while only a slight activity decrease was noticed at pH values between these two values. After elimination of excess NADPH the enzyme remained inactive for at least 4 h. The enzyme was protected against redox inactivation by low concentrations of GSSG, ferricyanide, GSH, or dithiothreitol, and high concentrations of NAD(P)+; oxidized glutathione effectively protected the enzyme at concentrations even lower than GSH. The inactive enzyme was efficiently reactivated after incubation with GSSG, ferricyanide, GSH, or dithiothreitol, whether NADPH was present or not. The reactivation with GSH was rapid even at 0 degree C, whereas the optimum temperature for reactivation with GSSG was 30 degrees C. A tentative model for the redox interconversion, involving an erroneous intramolecular disulfide bridge, is put forward.     

Mechanism of pigeon liver malic enzyme modification of histidyl residues by ethoxyformic anhydride.

Incubation of malic enzyme (L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) with ethoxyformic anhydride caused the time-dependent loss of its ability to catalyze reactions requiring the nucleotide cofactor NADP+ or NADPH, such as the oxidative decarboxylase, the NADP+ - stimualted oxalacetate decarboxylase, the pyruvate reductase, and the pyruvate-medium proton exchange activities. Similar loss of oxidative decarboxylase and pyruvate reductase activities was affected by photo-oxidation in the presence of rose bengal. The inactivation of oxidative decarboxylase activity by ethoxyformic anhydride was accompanied by the reaction of greater than or equal to 2.3 histidyl residues per enzyme site and was strongly inhibited by NADP+. Ethoxyformylation also impaired the ability of malic enzyme to bind NADP+ or NADPH. These results support the involvement of histidyl residue(s) at the nucleotide binding site of malic enzyme.     

Redox interconversion of Escherichia coli glutathione reductase. A study with permeabilized and intact cells

Summary The redox interconversion of Escherichia coli glutathione reductase has been studied both in situ, with permeabilized cells treated with different reductants, and in vivo, with intact cells incubated with compounds known to alter their intracellular redox state.The enzyme from toulene-permeabilized cells was inactivated in situ by NADPH, NADH, dithionite, dithiothreitol, or GSH. The enzyme remained, however, fully active upon incubation with the oxidized forms of such compounds. The inactivation was time-, temperature-, and concentration-dependent; a 50% inactivation was promoted by just 2 M NADPH, while 700 M NADH was required for a similar effect. The enzyme from permeabilized cells was completely protected against redox inactivation by GSSG, and to a lesser extent by dithiothreitol, GSH, and NAD(P)⁺. The inactive enzyme was efficiently reactivated in situ by physiological GSSG concentrations. A significant reactivation was promoted also by GSH, although at concentrations two orders of magnitude below its physiological concentrations. The glutathione reductase from intact E. coli cells was inactivated in vivo by incubation with DL-malate, DL-isocitrate, or higher L-lactate concentrations. The enzyme was protected against redox inactivation and fully reactivated by diamide in a concentration-dependent fashion. Diamide reactivation was not dependent on the synthesis of new protein, thus suggesting that the effect was really a true reactivation and not due to de novo synthesis of active enzyme. The glutathione reductase activity increased significantly after incubation of intact cells with tert-butyl or cumene hydroperoxides, suggesting that the enzyme was partially inactive within such cells. In conclusion, the above results show that both in situ and in vivo the glutathione reductase of Escherichia coli is subjected to a redox interconversion mechanism probably controlled by the intracellular NADPH and GSSG concentrations.     

The role of histidine residues in glutamate dehydrogenase

1. Glutamate dehydrogenase was subject to rapid inactivation when irradiated in the presence of Rose Bengal or incubated in the presence of ethoxyformic anhydride. 2. Inactivation in the presence of Rose Bengal led to the photo-oxidation of four histidine residues. Oxidation of three histidine residues had little effect on enzyme activity, but oxidation of the fourth residue led to the almost total loss of activity. 3. Acylation of glutamate dehydrogenase with ethoxyformic anhydride at pH6.1 led to the modification of three histidine residues with a corresponding loss of half the original activity. Acylation at pH7.5 led to the modification of two histidine residues and a total loss of enzyme activity. 4. One of the histidine residues undergoing reaction at pH6.1 also undergoes reaction at pH7.5. 5. The presence of either glutamate or NAD(+) in the reaction mixtures at pH6.1 had no appreciable effect. At pH7.5 glutamate caused a marked decrease in both the degree of alkylation and degree of inactivation. NAD(+) had no effect on the degree of inactivation at pH7.5 but did modify the extent of acylation. 6. The normal response of the enzyme towards ADP was unaffected by acylation at pH6.1 or 7.5. 7. The normal response of the enzyme towards GTP was altered by treatment at both pH6.1 and 7.5.     

Modification of lactate oxidase with diethyl pyrocarbonate. Evidence for an active-site histidine residue

1. Diethyl pyrocarbonate inactivated l-lactate oxidase from Mycobacterium smegmatis. 2. Two histidine residues underwent ethoxycarbonylation when the enzyme was treated with sufficient reagent to abolish more than 90% of the enzyme activity, but analyses of the inactivation showed that the modification of one histidine residue was sufficient to cause the loss of enzyme activity. The rates of enzyme inactivation and histidine modification were the same. 3. Substrate and competitive inhibitors decreased the maximum extent of inactivation to a 50% loss of enzyme activity and modification was decreased from 1.9 to 0.75–1.2 histidine residues modified/molecule of FMN. 4. Treatment of the enzyme with diethyl [¹⁴C]pyrocarbonate (labelled in the carbonyl groups) confirmed that only histidine residues were modified under the conditions used and that deacylation of the ethoxycarbonylhistidine residues by hydroxylamine was concomitant with the removal of the ¹⁴C label and the re-activation of the enzyme. 5. No evidence was found for modification of tryptophan, tyrosine or cysteine residues, and no difference was detected between the conformation and subunit structure of the modified and native enzyme. 6. Modification of the enzyme with diethyl pyrocarbonate did not alter the following properties: the binding of competitive inhibitors, bisulphite and substrate or the chemical reduction of the flavin group to the semiquinone or fully reduced states. The normal reduction of the flavin by lactate was, however, abolished.     

Glutathione reductase fromSaccharomyces cerevisiae undergoes redox interconversionin situ andin vivo

Redox interconversion of glutathione reductase was studiedin situ withS. cerevisiae. The enzyme was more sensitive to redox inactivation in 24 hour-starved cells than in freshly-grown ones. While 5 μM NADPH or 100 μM NADH caused 50% inactivation in normal cells in 30 min, 0.75 μM NADPH or 50 μM NADH promoted a similar effect in starved cells. GSSG reactivated the enzyme previously inactivated by NADPH, ascertaining that the enzyme was subjected to redox interconversion. Low EDTA concentrations fully protected the enzyme from NADPH inactivation, thus confirming the participation of metals in such a process. Extensive inactivation was obtained in permeabilized cells incubated with glucose-6-phosphate or 6-phosphogluconate, in agreement with the very high specific activities of the corresponding dehydrogenases. Some inactivation was also observed with malate, L-lactate, gluconate or isocitrate in the presence of low NADP⁺ concentrations. The inactivation of yeast glutathione reductase has also been studiedin vivo. The activity decreased to 75% after 2 hours of growth with glucono-δ-lactone as carbon source, while NADPH rose to 144% and NADP⁺ fell to 86% of their initial values. Greater changes were observed in the presence of 1.5 μM rotenone: enzymatic activity descended to 23% of the control value, while the NADH/NAD⁺ and NADPH/NADP⁺ ratios rose to 171% and 262% of their initial values, respectively. Such results indicate that the lowered redox potential of the pyridine nucleotide pool existing when glucono-δ-lactone is oxidized promotesin vivo inactivation of glutathione reductase.     

Mouse-liver glutathione reductase. Purification, kinetics, and regulation.

Glutathione reductase from the liver of DBA/2J mice was purified to homogeneity by means of ammonium sulfate fractionation and two subsequent affinity chromatography steps using 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose and N6-(6-aminohexyl)-adenosine 2',5'-biphosphate-Sephadex columns. A facile procedure for the synthesis of 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose is also presented. The purified enzyme exhibits a specific activity of 158 U/mg and an A280/A460 of 6.8. It was shown to be a dimer of Mr 105000 with a Stokes radius of 4.18 nm and an isoelectric point of 6.46. Amino acid composition revealed some similarity between the mouse and the human enzyme. Antibodies against mouse glutathione reductase were raised in rabbits and exhibited high specificity. The catalytic properties of mouse liver glutathione reductase have been studied under a variety of experimental conditions. As with the same enzyme from other sources, the kinetic data are consistent with a 'branched' mechanism. The enzyme was stabilized against thermal inactivation at 80 degrees C by GSSG and less markedly by NADP+ and GSH, but not by NADPH or FAD. Incubation of mouse glutathione reductase in the presence of NADPH or NADH, but not NADP+ or NAD+, produced an almost complete inactivation. The inactivation by NADPH was time, pH and concentration dependent. Oxidized glutathione protected the enzyme against inactivation, which could also be reversed by GSSG or other electron acceptors. The enzyme remained in the inactive state even after eliminating the excess NADPH. The inactive enzyme showed the same molecular weight as the active glutathione reductase. The spectral properties of the inactive enzyme have also been studied. It is proposed that auto-inactivation of glutathione reductase by NADPH and the protection as well as reactivation by GSSG play in vivo an important regulatory role.     

Affinity chromatography of glutathione reductase: bound by immobilized GSSG, eluted by NADPH.

Glutathione reductase bound to an affinity matrix of immobilized GSSG was eluted by its coenzyme NADPH rather than by its substrate GSSG or by NADH. NADP⁺ could also elute the enzyme, but a high concentration was needed to release enzyme activity in a sharp peak. This chromatographic system exhibits an unusual form of biospecificity in which the enzyme is bound to an immobilized substrate but released by its soluble cofactor.     

Reductive inactivation of yeast glutathione reductase by Fe(II) and NADPH

Glutathione reductase (GR) carries out the enzymatic reduction of glutathione disulfide (GSSG) to its reduced form (GSH) at the expense of the reducing power of NADPH. Previous studies have shown that GR from several species is progressively inactivated in the presence of NADPH, but that the mechanism of inactivation (especially in the presence of metals) has not been fully elucidated. We have investigated the involvement of iron ions in the inactivation of yeast (Saccharomyces cerevisiae) GR in the presence of NADPH. Even in the absence of added iron, inactivation of GR was partly blocked by the iron chelators, deferoxamine and ortho-phenanthroline, suggesting the involvement of trace amounts of contaminating iron in the mechanism of inhibition. Exogenously added antioxidants including ethanol, dimethylsulfoxide and 2-deoxyribose did not protect GR against NADPH-induced inactivation, whilst addition of exogenous Fe(II) (but not Fe(III)) potentiated the inactivation. Moreover, removal of oxygen from the medium led to increased inhibition of GR, whereas pre-incubation of the Fe(II)-containing medium for 30 min under normoxic conditions prior to the addition of GR abolished the enzyme inactivation by NADPH. Under these pre-incubation conditions, Fe(II) is fully oxidized to Fe(III) within 1 min. Furthermore, GR that had been previously inactivated in the presence of Fe(II) plus NADPH could be partially reactivated by treatment with ortho-phenanthroline and deferoxamine. In contrast, Fe(III) had no effect on GR reactivation. Together, these results indicate that yeast GR is inactivated by a reductive mechanism mediated by NADPH and Fe(II). According to this mechanism, GR is diverted from its normal redox cycling by the generation of an inactive reduced enzyme form in which both the FAD and thiol groups at the active site are likely in a reduced state.     

Copper-dependent inhibition and oxidative inactivation with affinity cleavage of yeast glutathione reductase

Effects of copper on the activity and oxidative inactivation of yeast glutathione reductase were analyzed. Glutathione reductase from yeast was inhibited by cupric ion and more potently by cuprous ion. Copper ion inhibited the enzyme noncompetitively with respect to the substrate GSSG and NADPH. The K_i values of the enzyme for Cu²⁺ and Cu⁺ ion were determined to be 1 and 0.35 μM, respectively. Copper-dependent inactivation of glutathione reductase was also analyzed. Hydrogen peroxide and copper/ascorbate also caused an inactivation with the cleavage of peptide bond of the enzyme. The inactivation/fragmentation of the enzyme was prevented by addition of catalase, suggesting that hydroxyl radical produced through the cuprous ion-dependent reduction of oxygen is responsible for the inactivation/fragmentation of the enzyme. SDS-PAGE and TOF–MS analysis confirmed eight fragments, which were further determined to result from the cleavage of the Met17-Ser18, Asn20-Thr21, Glu251-Gly252, Ser420-Pro421, Pro421-Thr422 bonds of the enzyme by amino-terminal sequencing analysis. Based on the kinetic analysis and no protective effect of the substrates, GSSG and NADPH on the copper-mediated inactivation/fragmentation of the enzyme, copper binds to the sites apart from the substrate-sites, causing the peptide cleavage by hydroxyl radical. Copper-dependent oxidative inactivation/fragmentation of glutathione reductase can explain the prooxidant properties of copper under the in vivo conditions.     

Effects of Exogenous Proline and Glycinebetaine on the Salt Tolerance of Rice Cultivars

Glutathione reductase was purified from iron-grown Thiobacillus ferrooxidas AP19-3 to an electrophoretically homogeneous state. The enzyme had an apparent molecular weight of 100,000 and was composed of two identical subunits of molecular weight (M_rs, 52,000) as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. A purified enzyme reduced one mole of the oxidized form of glutathione (GSSG) with one mole of NADPH to produce two moles of the reduced form of glutathione (GSH) and one mole of NADP⁺. The glutathione reductase was most active at pH 6.5 and 40°C, and had an isoelectric point at 5.1. The Michaelis constants of glutathione reductase for GSSG, NADPH, and NADH were 300, 26, and 125 μM, respectively.     

The redox interconversion mechanism of Saccharomyces cerevisiae glutathione reductase

The changes undergone by pure yeast glutathione reductase during redox interconversion have been studied. Both the active and inactive forms of the enzyme had similar molecular masses, suggesting that the inactivation is probably due to intramolecular modification(s). The glutathione reductase and transhydrogenase activities were similarly inactivated by NADPH and reactivated by GSH, while the diaphorase activity remained unaltered during redox interconversion of glutathione reductase. These results suggest that the inactivation site could be located far from the NADPH-binding site, although interfering with transhydrogenase activity, perhaps by conformational changes. The inactivation of glutathione reductase by 0.2 mM NADPH at pH 8 was paralleled by a gradual decrease in the absorbance at 530 nm and a simultaneous increase in the absorbance at 445 nm, while the reactivation promoted by GSH was initially associated with reversal of these spectral changes. The inactive enzyme spectrum retained some absorbance between 500 nm and 700 nm, showing a shoulder at 580-600 nm. Upon treatment of the enzyme with NADPH at pH 6.5 the spectrum remained unchanged, while no redox inactivation was observed under these conditions. It is suggested that the redox inactivation could be associated with the disappearance of the charge-transfer complex between the proximal thiolate and oxidized FAD in the two-electron-reduced enzyme. The inactive enzyme was reactivated by low GSSG concentrations, moderate dithiol concentrations, and high monothiol concentrations. These results and the spectral changes described above support the hypothesis attributing the redox interconversion to formation/disappearance of an erroneous disulfide between one of the half-cystines located at the GSSG-binding site and another cysteine nearby.     

Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography

Glutathione reductase (EC 1.6.4.2) was purified from spinach (Spinacia oleracea L.) leaves by affinity chromatography on ADP-Sepharose. The purified enzyme has a specific activity of 246 enzyme units/mg protein and is homogeneous by the criterion of polyacrylamide gel electrophoresis on native and SDS-gels. The enzyme has a molecular weight of 145,000 and consists of two subunits of similar size. The pH optimum of spinach glutathione reductase is 8.5–9.0, which is related to the function it performs in the chloroplast stroma. It is specific for oxidised glutathione (GSSG) but shows a low activity with NADH as electron donor. The pH optimum for NADH-dependent GSSG reduction is lower than that for NADPH-dependent reduction. The enzyme has a low affinity for reduced glutathione (GSH) and for NADP⁺, but GSH-dependent NADP⁺ reduction is stimulated by addition of dithiothreitol. Spinach glutathione reductase is inhibited on incubation with reagents that react with thiol groups, or with heavymetal ions such as Zn²⁺. GSSG protects the enzyme against inhibition but NADPH does not. Pre-incubation of the enzyme with NADPH decreases its activity, so kinetic studies were performed in which the reaction was initiated by adding NADPH or enzyme. The Km for GSSG was approximately 200 M and that for NADPH was about 3 M. NADP⁺ inhibited the enzyme, assayed in the direction of GSSG reduction, competitively with respect to NADPH and non-competitively with respect to GSSG. In contrast, GSH inhibited non-competitively with respect to both NADPH and GSSG. Illuminated chloroplasts, or chloroplasts kept in the dark, contain equal activities of glutathione reductase. The kinetic properties of the enzyme (listed above) suggest that GSH/GSSG ratios in chloroplasts will be very high under both light and dark conditions. This prediction was confirmed experimentally. GSH or GSSG play no part in the light-induced activation of chloroplast fructose diphosphatase or NADP⁺-glyceraldehyde-3-phosphate dehydrogenase. We suggest that GSH helps to stabilise chloroplast enzymes and may also play a role in removing H₂O₂. Glucose-6-phosphate dehydrogenase activity may be required in chloroplasts in the dark in order to provide NADPH for glutathione reductase.Abbreviations GSH reduced form of the tripeptide glutathione - GSSG oxidised form of glutathione     

Purification and characterization of the flavoenzyme glutathione reductase from rat liver.

Glutathione reductase from rat liver has been purified greater than 5000-fold in a yield of 20%. The molecular weights of the enzyme and its subunits were estimated to be 125,000 and 60,000, respectively, indicating that the native enzyme is a dimer. The enzyme molecular contains 2 FAD molecules, which are reducible by NADPH, GSH or dithioerythritol. The reduced flavin is instantaneously reoxidized by addition of GSSG. The steady state kinetic data are consistent with a branching reaction mechanism previously proposed for glutathione reductase from yeast (MANNERVIK, B. (1973) Biochem. Biophy. Res. Commun. 53, 1151-1158). This mechanism is also favored by the nonlinear inhibition pattern produced by NADP-+. However, at low GSSG concentrations the rate equation can be approximated by that of a simple ping pong mechanism. NADPH and the mixed disulfide of coenzyme A and GSH were about 10% as active as NADPH and GSSG, respectively, whereas some sulfenyl derivatives related to GSSG were less active as substrates. The pH activity profiles of these substrates differed from that of the NADPH-GSSG substrate pair.     

Chloroplast glutathione reductase: Purification and properties

Glutathione reductase was partially purified from isolated pea chloroplasts ( Pisum sativum L. cv. Progress #9). A 1600-fold purification was obtained and the purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)⁻¹ min⁻¹. The enzyme had a native molecular weight of approximately 156 kdalton and consisted of two each of two subunits of about 41 and 42 kdalton. The K_m for oxidized glutathione was 11 μ M and the K_m for NADPH was 1.7 μ M . Enzyme activity was affected by the ionic strength of the assay medium, and maximum activity was observed at an ionic strength of between 60 and 100 m M . The enzyme was inactivated by sulfhydryl modifying reagents and the presence of either oxidized glutathione or NADPH affected the extent of inactivation. Chloroplast glutathione reductase probably serves in the removal of photosynthetically derived H₂O₂ by reducing dehydroascorbate for ascorbate-linked reduction of H₂O₂. Intermediates of this reaction sequence, dehydroascorbate, ascorbate, reduced glutathione, and NADPH had no effect on enzymic activity.     

Interaction of NADP(H) with oxidized and reduced P450 reductase during catalysis. Studies with nucleotide analogues

Previous studies have shown that the interaction of P450 reductase with bound NADP(H) is essential to ensure fast electron transfer through the two flavin cofactors. In this study we investigated in detail the interaction of the house fly flavoprotein with NADP(H) and a number of nucleotide analogues. 1,4,5,6-Tetrahydro-NADP, an analogue of NADPH, was used to characterize the interaction of P450 reductase with the reduced nucleotide. This analogue is inactive as electron donor, but its binding affinity and rate constant of release are very close to those for NADPH. The 2'-phosphate contributes about 5 kcal/mol of the binding energy of NADP(H). Oxidized nicotinamide does not interact with the oxidized flavoprotein, while reduced nicotinamide contributes 1.3 kcal/mol of the binding energy. Oxidized P450 reductase binds NADPH with a K(d) of 0.3 microM, while the affinity of the reduced enzyme is considerably lower, K(d) = 1.9 microM. P450 reductase catalyzes a transhydrogenase reaction between NADPH and oxidized nucleotides, such as thionicotinamide-NADP(+), acetylpyridine-NADP(+), or [(3)H]NADP(+). The reverse reaction, reduction of [(3)H]NADP(+) by the reduced analogues, is also catalyzed by P450 reductase. We define the mechanism of the transhydrogenase reaction as follows: NADPH binding, hydride ion transfer, and release of the NADP(+) formed. An NADP(+) or its analogue binds to the two-electron-reduced flavoprotein, and the electron-transfer steps reverse to transfer hydride ion to the oxidized nucleotide, which is released. Measurements of the flavin semiquinone content, rate constant for NADPH release, and transhydrogenase turnover rates allowed us to estimate the steady-state distribution of P450 reductase species during catalysis, and to calculate equilibrium constants for the interconversion of catalytic intermediates. Our results demonstrate that equilibrium redox potentials of the flavin cofactors are not the sole factor governing rapid electron transfer during catalysis, but conformational changes must be considered to understand P450 reductase catalysis.     

Chemical modification of the histidine residue in phospholipase A2 (Naja naja naja). A case of half-site reactivity.

Reaction of phospholipase A2 (Naja naja naja) with p-bromophenacyl bromidine leads to almost complete loss of enzymatic activity. The rate of inactivation is pH-dependent with pKa equals 6.9 for the ionizing residue. p-Bromophenacyl bromide modifies 0.5 mol of histidine/mol of enzyme as judged by amino acid analysis and incorporation studies with 14C-labeled reagent. The rate of inactivation is affected by various cations; a saturating concentration of Ca2+ decreases the rate 5-fold, while Mn2+ increases the rate by a factor of 2. Triton X-100, which by itself has little affinity for the enzyme, protects against inactivation, presumably by sequestering p-bromophenacyl bromide into the apolar micellar core. The mixed micelle system of Triton X-100, dipalmitoyl phosphatidylcholine, and Ba2+ offers the best protection, lowering the inactivation rate by at least 50-fold. This suggests an active site role for the histidine residue. Ethoxyformic anhydride also modifies phospholipase A2, by acylation of the two amino groups, a tyrosine, and 0.5 mol of histidine/mol of enzyme without totally inactivating the enzyme. Removal of the ethoxyformyl group from the histidine does not reactivate the enzyme. Thus, modification of 0.5 mol of histidine with this reagent is not responsible for the 85% loss of activity seen. Ethoxyformylated enzyme, with 0.5 mol of acylated histidine/mol of enzyme, can be further inactivated by treatment with p-bromophenacyl bromide. The resulting derivative contains 0.4 mol of the 14C-labeled p-bromophenacyl group. Other modifiable groups do not show this half-residue reactivity. For example, oxidation of phospholipase A2 with N-bromosuccinimide leads to rapid destruction of 1.0 tryptophan residue and 5% residual activity. The results of these chemical modification experiments can be interpreted in terms of a model in which the active species of enzyme interacting with mixed micelles is a dimer (or possibly higher order aggregate). The dimer, though composed of identical subunits, is asymmetric; the histidine of one subunit is accessible to ethoxyformic anhydride, while the other histidine is near a hydrophobic region of the enzyme and is chemically reactive toward p-bromophenacyl bromide.