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ABSTRACT: NADP(H) binding is essential for fast electron transfer through the flavoprotein domain of the fusion protein P450BM3. Here we characterize the interaction of NADP(H) with the oxidized and partially reduced enzyme and the effect of this interaction on the redox properties of flavin cofactors and electron transfer. Measurements by three different approaches demonstrated a relatively low affinity of oxidized P450BM3 for NADP(+), with a K(d) of about 10 microM. NADPH binding is also relatively weak (K(d) approximately 10 microM), but the affinity increases manyfold upon hydride ion transfer so that the active 2-electron reduced enzyme binds NADP(+) with a K(d) in the submicromolar range. NADP(H) binding induces conformational changes of the protein as demonstrated by tryptophan fluorescence quenching. Fluorescence quenching indicated preferential binding of NADPH by oxidized P450BM3, while no catalytically competent binding with reduced P450BM3 could be detected. The hydride ion transfer step, as well as the interflavin electron transfer steps, is readily reversible, as demonstrated by a hydride ion exchange (transhydrogenase) reaction between NADPH and NADP(+) or their analogues. Experiments with FMN-free mutants demonstrated that FAD is the only flavin cofactor required for the transhydrogenase activity. The equilibrium constants of each electron transfer step of the flavoprotein domain during catalytic turnover have been calculated. The values obtained differ from those calculated from equilibrium redox potentials by as much as 2 orders of magnitude. The differences result from the enzyme's interaction with NADP(H).
Biochemistry 11/2000; 39(41):12699-707. · 3.42 Impact Factor
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ABSTRACT: 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.
Biochemistry 06/2000; 39(17):5066-74. · 3.42 Impact Factor
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ABSTRACT: The interaction of recombinant house fly (Musca domestica) P450 reductase with NADPH and the role of the FMN semiquinone in reducing cytochrome c have been investigated. House fly P450 reductase can rapidly oxidize only one molecule of NADPH, whereas the rate of oxidation of a second molecule of NADPH is too slow to account for the observed rates of catalysis. This demonstrates that house fly P450 reductase does not require a priming reaction with NADPH for catalysis. Kinetics of cytochrome c reduction and EPR spectroscopy revealed that the enzyme forms two types of neutral FMN semiquinone. One serves as the catalytic intermediate of cytochrome c reduction, and another one is an 'airstable' semiquinone, which reduces cytochrome c 3000 times more slowly. The results show that the reduction state of the house fly P450 reductase during catalysis cycles in a 0-2-1-0 sequence.
FEBS Letters 07/1999; 453(1-2):201-4. · 3.54 Impact Factor
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ABSTRACT: Recombinant house fly (Musca domestica) cytochrome P450 reductase has been purified by anion exchange and affinity chromatography. Steady-state kinetics of cytochrome c reductase activity revealed a random Bi-Bi mechanism with formation of a ternary P450 reductase-NADPH-electron acceptor complex as catalytic intermediate. NADP(H) binding is essential for fast hydride ion transfer to FAD, as well as for electron transfer from FMN to cytochrome c. Reduced cytochrome c had no effect on the enzyme activity, while NADP+ and 2'-AMP inhibited P450 reductase competitively with respect to NADPH and noncompetitively with respect to cytochrome c. The affinity of the P450 reductase to NADPH is 10 times higher than to NADP+ (Kd of 0.31 and 3.3 microM, respectively). Such an affinity change during catalysis could account for a +30 mV shift of the redox potential of FAD. Cys560 was substituted for Tyr by site-directed mutagenesis. This mutation decreased enzyme affinity to NADPH 35-fold by decreasing the bimolecular rate constant of nucleotide binding with no detectable effect on the kinetic mechanism. The affinity of the C560Y mutant enzyme to NADP+ decreased 9-fold compared to the wild-type enzyme, while the affinity to 2'-AMP was not significantly affected, suggesting that Cys560 is located in the nicotinamide binding site of the active, full-size enzyme in solution.
Insect Biochemistry and Molecular Biology 04/1999; 29(3):233-42. · 3.25 Impact Factor
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M B Murataliev
Methods in molecular biology (Clifton, N.J.) 02/1999; 131:97-110.
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ABSTRACT: A cDNA encoding a cytochrome P450 enzyme was isolated from a cDNA library of the corpora allata (CA) from reproductively active Diploptera punctata cockroaches. This P450 from the endocrine glands that produce the insect juvenile hormone (JH) is most closely related to P450 proteins of family 4 and was named CYP4C7. The CYP4C7 gene is expressed selectively in the CA; its message could not be detected in the fat body, corpora cardiaca, or brain, but trace levels of expression were found in the midgut and caeca. The levels of CYP4C7 mRNA in the CA, measured by ribonuclease protection assays, were linked to the activity cycle of the glands. In adult females, CYP4C7 expression increased immediately after the peak of JH synthesis, reaching a maximum on day 7, just before oviposition. mRNA levels then declined after oviposition and during pregnancy. The CYP4C7 protein was produced in Escherichia coli as a C-terminal His-tagged recombinant protein. In a reconstituted system with insect NADPH cytochrome P450 reductase, cytochrome b5, and NADPH, the purified CYP4C7 metabolized (2E,6E)-farnesol to a more polar product that was identified by GC-MS and by NMR as (10E)-12-hydroxyfarnesol. CYP4C7 converted JH III to 12-trans-hydroxy JH III and metabolized other JH-like sesquiterpenoids as well. This omega-hydroxylation of sesquiterpenoids appears to be a metabolic pathway in the corpora allata that may play a role in the suppression of JH biosynthesis at the end of the gonotrophic cycle.
Proceedings of the National Academy of Sciences 11/1998; 95(22):12884-9. · 9.68 Impact Factor
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ABSTRACT: Experiments are reported on the uni-site catalysis and the transition from uni-site to multi-site catalysis with bovine heart mitochondrial F1-ATPase. The very slow uni-site ATP hydrolysis is shown to occur without tightly bound nucleotides present and with or without Pi in the buffer. Measurements of the transition to higher rates and the amount of bound ATP committed to hydrolysis as the ATP concentration is increased at different fixed enzyme concentrations give evidence that the filling of a second site can initiate near maximal turnover rates. They provide rate constant information, and show that an apparent Km for a second site of about 2 microM and Vmax of 10 s-1, as suggested by others, is not operative. Careful initial velocity measurements also eliminate other suggested Km values and are consistent with bi-site activation to near maximal hydrolysis rates, with a Km of about 130 microM and Vmax of about 700 s-1. However, the results do not eliminate the possibility of additional 'hidden' Km values with similar Vmax:Km ratios. Recent data on competition between TNP-ATP and ATP revealed a third catalytic site for ATP in the millimolar concentration range. This result, and those reported in the present paper, allow the conclusion that the mitochondrial F1-ATPase can attain near maximal activity in bi-site catalysis. Our data also add to the evidence that a recent claim, that the mitochondrial F1-ATPase does not show catalytic site cooperativity, is invalid.
Biochemical Journal 04/1998; 330 ( Pt 2):1037-43. · 4.90 Impact Factor
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ABSTRACT: Cytochrome P450BM3 is a self-sufficient soluble fatty acid hydroxylase from Bacillus megaterium utilizing tightly bound FAD and FMN cofactors to transfer reducing equivalents from NADPH to the heme active site. Active-inactive transitions of cytochrome P450BM3 were exploited to identify catalytic intermediates of the enzyme. Shortly upon reduction by NADPH, a two-electron reduced active P450BM3 is formed with two flavin semiquinones, anionic and neutral, present simultaneously. P450BM3 inactivated by NADPH has a three-electron reduced flavoprotein domain. NADPH is unable to reduce P450BM3 rapidly unless the flavoprotein domain is fully oxidized. During steady-state hydroxylation of a poor substrate, tetradecanol, the flavoprotein reduction state does not exceed two, with two flavin semiquinones, anionic and neutral, present. Absorbance and EPR spectroscopic characterization of both anionic and neutral flavin semiquinone is presented. NADPH and NADH were compared as electron donors for P450BM3-catalyzed fatty acid hydroxylation and cytochrome c and heme iron reduction. The Km for NADH of 3-5 mM is about 3000 times higher than the Km of 1-1.5 microM for NADPH. Although NADH can support cytochrome c reduction and fatty acid hydroxylation with the rates as high as 22 and 13 s-1, respectively, these turnover numbers are only about 20% of those observed with NADPH. The results suggest that nucleotide binding plays an important role in catalysis by controlling electron-transfer properties of the flavin cofactors. In W574G and G570D mutant P450BM3 enzymes that are deficient in FMN, NADP+ binding stabilizes fully reduced FAD. P450BM3 catalyzes single-turnover and steady-state laurate hydroxylation with near stoichiometric product formation at NADPH concentrations below that of the enzyme. A mechanism of electron transfer by the flavoprotein domain of P450BM3 is proposed with the reduction state of the flavoprotein domain cycling in a 0-2-1-0 sequence. We also propose that an interaction of bound NADP+ with anionic FAD semiquinone is essential for splitting a pair of electrons that are then transferred in two one-electron transfer steps to the heme catalytic site.
Biochemistry 08/1997; 36(27):8401-12. · 3.42 Impact Factor
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ABSTRACT: P450BM3 is a bacterial fusion protein between a cytochrome P450 fatty acid hydroxylase (CYP102) and an FAD- and FMN-containing flavoprotein homologous to NADPH: cytochrome P450 reductase. It has been shown that incubation of P450BM3 with NADPH in the absence of a fatty acid substrate results in inhibition of hydroxylase activity [Narhi, L. O., & Fulco, A. J. (1986) J. Biol. Chem. 261, 7160-7169]. We show that laurate-dependent oxidation of NADPH and oxygen consumption are also inhibited under those conditions. The inhibited enzyme is unable to transfer electrons to the heme iron, but reduces artificial electron acceptors such as cytochrome c, 2,6-dichlorophenolindophenol, or ferricyanide. Incubation with these acceptors rapidly restores hydroxylase activity of P450BM3. The active enzyme is able to catalyze the reduction of cytochrome c and hydroxylation of laurate simultaneously. Cytochrome c has no effect on the K(m) and Vmax of laurate hydroxylation. Laurate and other substrates stimulate cytochrome c reduction by 50-70%. Carbon monoxide inhibits hydroxylase activity, but stimulates cytochrome c reduction 3-4 fold and has no effect on the K(m) for cytochrome c. This stimulation requires binding of a substrate at the heme catalytic site. Laurate binding induces conformational changes in the flavoprotein domain as shown by a 2-fold increase of the flavin fluorescence. Inactivation of P450BM3 by NADPH abolishes the stimulation of cytochrome c reduction by laurate and CO. Complete inhibition of hydroxylase activity correlates with complete lack of stimulation of cytochrome c reduction. The results suggest that a specific conformation of the two domains is maintained in the active P450BM3, ensuring high hydroxylase activity. Cytochrome c reductase and hydroxylase activities of P450BM3 involve different sites of interaction with the flavoprotein domain, different catalytic intermediates, and different rate-limiting steps.
Biochemistry 11/1996; 35(47):15029-37. · 3.42 Impact Factor
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ABSTRACT: A microsomal cytochrome b5 cDNA from the house fly, Musca domestica, was cloned and sequenced. The deduced amino acid sequence of the full-length house fly cytochrome b5 (134 residues) is 48% identical to that of rat microsomal cytochrome b5. The house fly cytochrome b5 protein was overexpressed in Escherichia coli, purified, and characterized. Absorption and EPR spectroscopy reveal properties very similar to cytochromes b5 from vertebrates. NMR spectra indicate that the orientation of the heme in the protein relative to its alpha,gamma meso axis is about 1:1. A redox potential of -26 mV versus standard hydrogen electrode was measured by cyclic voltammetry on a modified gold electrode in the presence of hexamminechromium(III) chloride. The cytochrome b5 is reduced by house fly cytochrome P450 reductase in a reconstituted system at a high rate (5.5 s-1), and it stimulates heptachlor epoxidation when reconstituted with house fly cytochrome P450 reductase, cytochrome P450 6A1, phospholipid, and detergent. Cytochrome b5 decreases the apparent Km for P450 reductase and increases the Vmax for heptachlor epoxidation at constant cytochrome P450 6A1 concentrations. The results indicate that cytochrome b5 stimulates a step following the first electron transfer during cytochrome P450 6A1 turnover.
Journal of Biological Chemistry 11/1996; 271(43):26637-45. · 4.77 Impact Factor
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M B Murataliev
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ABSTRACT: It was shown recently that ATP present at near saturating concentrations did not prevent binding and hydrolysis of submicromolar concentration of trinitrophenyl adenosine triphosphate (Tnp-ATP) by F1-ATPase [Murataliev, M. B. & Boyer, P. O. (1994) J. Biol. Chem. 269, 15431-15439]. To explore F1-ATPase binding sites that bind Tnp-ATP a new photoreactive analog of ATP, 2-azido-trinitrophenyl adenosine triphosphate (2-N3-Tnp-ATP) has been synthesized and used for photoaffinity labeling of mitochondrial F1-ATPase. The analog shares many properties of the parent non-azido Tnp-ATP as shown from spectral characteristics, binding with F1-ATPase, and kinetic and inhibition studies. 500 microM ATP does not prevent binding and hydrolysis of low concentrations of 2-N3-Tnp-ATP by F1-ATPase. Photoirradiation of the enzyme-analog complex formed under such conditions results in the labeling of the catalytic-site peptide. This shows that in the presence of near saturating ATP, Tnp-ATP can enter the catalytic cycle and inhibit ATP hydrolysis by initial binding at a third catalytic site. The results give strong evidence that only two catalytic sites need to have bound substrate for near maximal turnover rate, and that three catalytic sites of F1-ATPase participate equally in catalysis. When F1-ATPase binds substoichiometric 2-N3-Tnp-ATP in the presence of Mg2+, illumination of the inactive complex formed results in the covalent labeling of a catalytic site. This shows that F1-ATPase forms similar inactive complexes when ADP or Tnp-ADP is bound at a catalytic site in the presence of Mg2+. Exposure of the nucleotide-depleted F1-ATPase to 20 microM 2-N3-Tnp-ATP followed by a short incubation with excess of Tnp-ATP results in binding, and, upon illumination, in a covalent labeling of a non-catalytic-site peptide.
European Journal of Biochemistry 09/1995; 232(2):578-85. · 3.58 Impact Factor
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ABSTRACT: Relatively high ATP concentrations show an unexpected lack of inhibition of the hydrolysis of low concentrations of trinitrophenyl ATP (TNP-ATP) by mitochondrial F1-ATPase. In striking contrast low TNP-ATP concentrations markedly inhibit the hydrolysis of much higher ATP concentrations. The three catalytic sites undergoing sequential conformational changes have different conformations at any instant of catalysis, and only two need to be filled for rapid, steady-state ATP hydrolysis. The remaining site has low affinity for ATP (Kd 2 mM) but about 10(4) greater affinity for TNP-ATP (Km and Kd about 0.2 microM). Thus 500 microM ATP does not prevent binding of less than 1 microM TNP-ATP. As the site binding the TNP-ATP undergoes sequential conformational changes the TNP-ATP undergoes sequential conformational changes the TNP-ATP is hydrolyzed and products are released. The results give strong support to the view that all three catalytic sites proceed equivalently in ATP as well as TNP-ATP hydrolysis. The conformation that has the lowest affinity for ATP has over a 10-fold greater affinity for ADP (Kd 150 microM) and may be akin to the conformation to which ADP binds during net ATP synthesis by the ATP synthase. The recognition of these features was made possible by new information obtained from detailed studies of the interactions of Mg2+, TNP-ADP, TNP-ATP, ATP, and noncatalytic sites on initial and steady-state hydrolysis rates.
Journal of Biological Chemistry 07/1994; 269(22):15431-9. · 4.77 Impact Factor
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M B Murataliev
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ABSTRACT: The evidence is presented that the ADP- and Mg(2+)-dependent inactivation of MF1-ATPase during MgATP hydrolysis requires binding of ATP at two binding sites: one is catalytic and the second is noncatalytic. Binding of the noncatalytic ATP increases the rate of the inactive complex formation in the course of ATP hydrolysis. The rate of the enzyme inactivation during ATP hydrolysis depends on the medium Mg2+ concentration. High Mg2+ inhibits the steady-state activity of MF1-ATPase by increasing the rate of formation of inactive enzyme-ADP-Mg2+ complex, thereby shifting the equilibrium between active and inactive enzyme forms. The Mg2+ needed for MF1-ATPase inactivation binds from the medium independent from the MgATP binding at either catalytic or noncatalytic sites. The inhibitory ADP molecule arises at the MF1-ATPase catalytic site as a result of MgATP hydrolysis. Exposure of the native MF1-ATPase with bound ADP at a catalytic site to 1 mM Mg2+ prior to assay inactivates the enzymes with kinact 24 min-1. The maximal inactivation rate during ATP hydrolysis at saturating MgATP and Mg2+ does not exceed 10 min-1. The results show that the rate-limiting step of the MF1-ATPase inactivation during ATP hydrolysis with excess Mg2+ precedes binding of Mg2+ and likely is the rate of formation of enzyme with ADP bound at the catalytic site without bound P(i). This complex binds Mg2+ resulting in inactive MF1-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)
Biochemistry 01/1993; 31(51):12885-92. · 3.42 Impact Factor
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ABSTRACT: The presence of ATP at non-catalytic sites of the chloroplast F1-ATPase (CF1) eliminates a considerable lag in onset of enzyme activity that otherwise occurs in the presence of bicarbonate [Milgrom, Y. M., Ehler, L. & Boyer, P. D. (1991) J. Biol. Chem. 266, 11551-11558]. Sulfite is known to be much more effective than bicarbonate in stimulating ATPase activity CF1. Results reported here show that when assayed in the presence of sulfite, CF1, with some non-catalytic sites empty or filled with GT(D)P, is able to hydrolyze both ATP and GTP. Thus, the presence of adenine nucleotides at non-catalytic sites is not necessary for catalytic turnover of CF1. However, even though CF1 with empty non-catalytic sites shows a significant initial activity, the prior binding of adenine nucleotides at non-catalytic site(s) results in further activation of MgATPase and MgGTPase activities, even at relatively high sulfite and substrate concentrations. Although extensive activation of CF1 results from the presence of sulfite, with or without nucleotide binding at non-catalytic sites, the Km remains constant, at about 50 microM for MgATP and 400 microM for MgGTP. The results obtained show that the ATPase activity of CF1 is determined by the fraction of the active enzyme. The inactive CF1.ADP.Mg2+ formed during MgATP hydrolysis can be rapidly trapped by azide to provide a measure of the fraction of inactive enzyme. Increasing the concentration of sulfite increases the fraction of active CF1 in the assay medium. Measurements with radioactively labeled nucleotides show that the presence of ATP at non-catalytic sites promotes the ATP-dependent release of inhibitory ADP from a catalytic site. The activating effect of ATP binding at non-catalytic sites results from increasing the portion of CF1 in an active state during steady-state ATP hydrolysis.
European Journal of Biochemistry 10/1992; 209(2):681-7. · 3.58 Impact Factor
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ABSTRACT: The interactions between ADP, Mg2+, and azide that result in the inhibition of the chloroplast F1 ATPase (CF1) have been explored further. The binding of the inhibitory Mg2+ with low Kd is shown to occur only when tightly bound ADP is present at a catalytic site. Either the tightly bound ADP forms part of the Mg(2+)-binding site or it induces conformational changes creating the high-affinity site for inhibitory Mg2+. Kinetic studies show that CF1 forms two catalytically inactive complexes with Mg2+. The first complex results from Mg2+ binding with a Kd for Mg2+ dissociation of about 10-15 microM, followed by a slow conversion to a complex with a Kd of about 4 microM. The rate-limiting step of the CF1 inactivation by Mg2+ is the initial Mg2+ binding. When medium Mg2+ is chelated with EDTA, the two complexes dissociate with half-times of about 1 and 7 min, respectively. Azide enhances the extent of Mg(2+)-dependent inactivation by increasing the affinity of the enzyme for Mg2+ 3-4 times and prevents the reactivation of both complexes of CF1 with ADP and Mg2+. This results from decreasing the rate of Mg2+ release; neither the rate of Mg2+ binding to CF1 nor the rate of isomerization of the first inactive complex to the more stable form is affected by azide. This suggests that the tight-binding site for the inhibitory azide requires prior binding of both ADP and Mg2+.
Biochemistry 09/1991; 30(34):8305-10. · 3.42 Impact Factor
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ABSTRACT: Interaction of F1-ATPase from beef heart mitochondria with PPi has been investigated. The presence of PPi in the ATPase assay medium does not affect the initial rate of ATP hydrolysis by F1-ATPase, but slows down the decrease of enzyme activity in the course of ATP hydrolysis and increases the steady-state rate of ATP hydrolysis. Being present in the ATPase assay medium, PPi accelerates the ATP-dependent reactivation of an inactive complex formed by F1-ATPase and ADP. This inactive complex is also reactivated after preincubation with PPi. F1-ATPase, preincubated with PPi, is inactivated by azide much more slowly than is the non-preincubated enzyme. PPi stimulates the binding of Pi to F1-ATPase by decreasing mainly the Kd for Pi and only slightly raising the stoichiometry of high-affinity Pi binding. It follows from the results obtained that PPi interacts with the non-catalytic site(s) of F1-ATPase.
European Journal of Biochemistry 11/1988; 177(1):213-8. · 3.58 Impact Factor
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ABSTRACT: Under conditions of molar excess of enzyme, isolated F1-ATPase from beef heart mitochondria catalyses ATP hydrolysis biphasically. The rate constants for product release are approximately 10(-1) and 10(-4)-10(-3) s-1, respectively. The slow phase of ATP hydrolysis is insensitive to EDTA. [gamma-32P]ATP splitting in the slow phase cannot be chased from the enzyme during several catalytic turnovers. It follows from these results that the slow single-site hydrolysis of ATP (kcat approximately 10(-4) s-1), initially observed by Grubmeyer et al. [(1982) J. Biol. Chem. 257, 12092-12100], is not carried out by the normal catalytic site. In contrast, the phase of rapid ATP hydrolysis (kcat approximately 10(-1) s-1) is completely prevented by EDTA and is believed to be the normal function of the normal catalytic site of F1-ATPase.
FEBS Letters 10/1987; 222(1):32-6. · 3.54 Impact Factor
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ABSTRACT: The properties of the nucleotides tightly bound with mitochondrial F1-ATPase were examined. One of three bound nucleotide molecules is localized at the site with Kd approximately 10(-7) M and released with koff approximately 0.1 s-1. The second nucleotide molecule is bound with the enzyme with Kd approximately 10(-8) M and koff for its dissociation is 3 X 10(-4) s-1. The third is never released even in the presence of 1 mM ATP or ADP. The last two nucleotides are believed to be bound at the noncatalytic sites of F1-ATPase. Pyrophosphate promotes liberation of two releasable nucleotide molecules, decreasing the affinity of the enzyme to AD(T)P. From the results obtained it follows that the only suitable criterion for localization of the nucleotide at the F1-ATPase catalytic site is the high rate (koff greater than or equal to 0.1 s-1) of its spontaneous release.
FEBS Letters 08/1987; 219(1):156-60. · 3.54 Impact Factor
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ABSTRACT: Under the conditions of ATP regeneration and molar excess of nucleotide-depleted F1-ATPase the enzyme catalyses steady-state ATP hydrolysis by the single catalytic site. Values of Km = 10(-8) M and Vm = 0.05 s-1 for the single-site catalysis have been determined. ADP release limits single-site ATP hydrolysis under steady-state conditions. The equilibrium constant for ATP hydrolysis at the F1-ATPase catalytic site is less than or equal to 0.7.
FEBS Letters 03/1987; 212(1):63-7. · 3.54 Impact Factor
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ABSTRACT: The effect of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC) on the reactions catalyzed by the glucose-6-phosphatase system of rat liver microsomes was studied. Modification of the intact microsomes by CMC leads to the inhibition of the glucose-6-phosphatase, pyrophosphate:glucose and carbamoyl-phosphate : glucose phosphotransferase activities of the system. The activities are restored by the disruption of the microsomal permeability barrier. The mannose-6-phosphate, pyrophosphate, and carbamoyl-phosphate phosphohydrolase activities of the intact as well as the disrupted microsomes were not affected by CMC. It follows from the results obtained that CMC inactivates the microsomal glucose-6-phosphate translocase, the inactivation is a result of the modification of a single sulfhydryl or amino group of the translocase; pyrophosphate, carbamoyl phosphate and inorganic phosphate are transported across the microsomal membrane without participation of the glucose-6-phosphate translocase; pyrophosphate and carbamoyl phosphate may act as the phosphate donors in the glucose phosphorylation reactions in vivo.
European Journal of Biochemistry 11/1986; 160(2):401-5. · 3.58 Impact Factor
