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1.
Using guanidinium and n-butylammonium cations (C+) as models for the positively charged side chains in arginine and lysine, we have determined the association constants with various oxyanions by potentiometric titration. For a dibasic acid, H2A, three association complexes may exist: K1M = [CHA][C+] [HA?]; K1D = [CA?][C+] [A2?]; K2D = [C2A][C+] [CA?]. For guanidinium ion and phosphate, K1M = 1.4, K1D = 2.6, and K2D = 5.1. The data for carboxylates indicate that the basicity of the oxyanion does not affect the association constant: acetate, pKa = 4.8, K1M = 0.37; formate, pKa = 3.8, K1M = 0.32; and chloroacetate, pKa = 2.9, K1M = 0.43, all with guanidinium ion. Association constants are also reported for carbonate, dimethylphosphinate, benzylphosphonate, and adenylate anions.  相似文献   

2.
The observed equilibrium constants (Kobs) for the l-phosphoserine phosphatase reaction [EC 3.1.3.3] have been determined under physiological conditions of temperature (38 °C) and ionic strength (0.25 m) and physiological ranges of pH and free [Mg2+]. Using Σ and square brackets to indicate total concentrations Kobs = Σ L-serine][Σ Pi]Σ L-phosphoserine]H2O], K = L-H · serine±]HPO42?][L-H · phosphoserine2?]H2O]. The value of Kobs has been found to be relatively sensitive to pH. At 38 °C, K+] = 0.2 m and free [Mg2+] = 0; Kobs = 80.6 m at pH 6.5, 52.7 m at pH 7.0 [ΔGobs0 = ?10.2 kJ/mol (?2.45 kcal/mol)], and 44.0 m at pH 8.0 ([H2O] = 1). The effect of the free [Mg2+] on Kobs was relatively slight; at pH 7.0 ([K+] = 0.2 m) Kobs = 52.0 m at free [Mg2+] = 10?3, m and 47.8 m at free [Mg2+] = 10?2, m. Kobs was insignificantly affected by variations in ionic strength (0.12–1.0 m) or temperature (4–43 °C) at pH 7.0. The value of K at 38 °C and I = 0.25 m has been calculated to be 34.2 ± 0.5 m [ΔGobs0 = ?9.12 kJ/mol (?2.18 kcal/ mol)]([H2O] = 1). The K for the phosphoserine phosphatase reaction has been combined with the K for the reaction of inorganic pyrophosphatase [EC 3.6.1.1] previously estimated under the same physiological conditions to calculate a value of 2.04 × 104, m [ΔGobs0 = ?28.0 kJ/mol (?6.69 kcal/mol)] for the K of the pyrophosphate:l-serine phosphotransferase [EC 2.7.1.80] reaction. Kobs = [Σ L-serine][Σ Pi][Σ L-phosphoserine][H2O], K = [L-H · serine±]HPO42?][L-H · phosphoserine2?]H2O. Values of Kobs for this reaction at 38 °C, pH 7.0, and I = 0.25 m are very sensitive to the free [Mg2+], being calculated to be 668 [ΔGobs0 = ?16.8 kJ/mol (?4.02 kcal/mol)] at free [Mg2+] = 0; 111 [ΔGobs0 = ?12.2 kJ/mol (?2.91 kcal/mol)] at free [Mg2+] = 10?3, m; and 9.1 [ΔGobs0 = ?5.7 kJ/mol (?1.4 kcal/mol) at free [Mg2+] = 10?2, m). Kobs for this reaction is also sensitive to pH. At pH 8.0 the corresponding values of Kobs are 4000 [ΔGobs0 = ?21.4 kJ/mol (?5.12 kcal/mol)] at free [Mg2+] = 0; and 97.4 [ΔGobs0 = ?11.8 kJ/ mol (?2.83 kcal/mol)] at free [Mg2+] = 10?3, m. Combining Kobs for the l-phosphoserine phosphatase reaction with Kobs for the reactions of d-3-phosphoglycerate dehydrogenase [EC 1.1.1.95] and l-phosphoserine aminotransferase [EC 2.6.1.52] previously determined under the same physiological conditions has allowed the calculation of Kobs for the overall biosynthesis of l-serine from d-3-phosphoglycerate. Kobs = [Σ L-serine][Σ NADH][Σ Pi][Σ α-ketoglutarate][Σ d-3-phosphoglycerate][Σ NAD+][Σ L-glutamat0] The value of Kobs for these combined reactions at 38 °C, pH 7.0, and I = 0.25 m (K+ as the monovalent cation) is 1.34 × 10?2, m at free [Mg2+] = 0 and 1.27 × 10?2, m at free [Mg2+] = 10?3, m.  相似文献   

3.
The pH dependence of the reaction of tris(hydroxymethyl)aminomethane (Tris) with the activated carbonyl compound 4-trans-benzylidene-2-phenyloxazolin-5-one (I) is given by the equation k′2 = kbKa(Ka + [H+]) + ka[OH?]Ka(Ka + [H+]), where Ka is the dissociation constant of TrisH+. Spectrophotometric experiments show that the Tris ester of α-benzamido-trans-cinnamic acid is formed quantitatively over a range of pH values, regardless of the relative contribution of kb and ka terms to k2. Hence, both terms refer to alcoholysis. While the mechanism of the reaction is not determined unequivocally in the present work, the magnitude of the kb term, together with its dependence on the basic form of Tris, suggests that ester formation is occurring by nucleophilic attack of a Tris hydroxyl group on the carbonyl carbon of the oxazolinone, with intramolecular catalysis by the Tris amino group. The rate enhancement due to this group is at least 102 and possibly of the order 106. This system is compared with other model systems for the acylation step of catalysis by serine esterases and proteinases.  相似文献   

4.
N-Phenylhydroxylamine is oxidized in aqueous phosphate buffer to nitrosobenzene, nitrobenzene, and azoxybenzene. Degradation is O2 dependent and shows general catalysis by H2PO4? (k1 = 2.3 M?2 sec?1) and PO4?3 (k2 = 2.3 × 105M?2 sec?1) or kinetically equivalent terms. Evidence is presented suggesting the intermediacy of a highly reactive species leading to these products.  相似文献   

5.
The rates of electron exchange between ferricytochrome c (CIII)3 and ferrocytochrome c (CII) were observed as a function of the concentrations of ferrihexacyanide (FeIII) and ferrohexacyanide (FeII) by monitoring the line widths of several proton resonances of the protein. Addition of FeII to CIII homogeneously increased the line widths of the two downfield paramagnetically shifted heme methyl proton resonances to a maximal value. This was interpreted as indicating the formation of a stoichiometric complex, CIII·FeII, in the over-all reaction:
CIII+FeII?k?1k1CIII·FeII?k?2k2CII·FeIII?k?3k3CIII+FeII
Values for k1k?1 = 0.4 × 103m?1and k2 = 208 s?1, respectively, were calculated from the maximal change in line width observed at pH 7.0 and 25 °C. Changes in the line width of CIII in the presence of FeII and either KCl or FeIII suggest that complexation is principally ionic, that FeIII and FeII compete for a common site. Addition of saturating concentrations of FeIII to CIII produced only minor changes in the nuclear magnetic resonance spectrum of CIII suggesting that complexation occurs on the protein surface.Addition of FeIII to CII in the presence of excess FeII (to retain most of the protein as CII) increased the line width of the methyl protons of ligated methionine 80. A value for k?2 ≈ 2.08 × 104 s?1 was calculated from the dependence of linewidth on the concentration of FeII at 24 °C. These rates are shown to be consistent with the over-all rates of reduction and oxidation previously determined by stopped flow measurements, indicating that k2 and k?2 were rate limiting. From the temperature dependence the enthalpies of activation are 7.9 and 15.2 kcal/mol for k2 and k?2, respectively.  相似文献   

6.
Presteady-state kinetic studies of α-chymotrypsin-catalyzed hydrolysis of a specific chromophoric substrate, N-(2-furyl)acryloyl-l-tryptophan methyl ester, were performed by using a stopped-flow apparatus both under [E]0 ? [S]0 and [S]0 ? [E]0 conditions in the pH range of 5–9, at 25 °C. The results were accounted for in terms of the three-step mechanism involving enzyme-substrate complex (E · S) and acylated enzyme (ES′); no other intermediate was observed. This substrate was shown to react very efficiently, i.e., the maximum of the second-order acylation rate constant (k2Ks)max = 4.2 × 107 M?1 s?1. The limiting values of Ks′ (dissociation constant of E · S), K2 (acylation rate) and k3 (deacylation rate) were obtained from the pH profiles of these parameters to be 0.6 ± 0.2 × 10?5 m, 360 ± 15 s?1 and 29.3 ± 0.8 s?1, respectively. Likewise small values were observed for Ki of N-(2-furyl)-acryloyl-l-tryptophan and N-(2-furyl)acryloyl-d-tryptophan methyl ester and Km of N-(2-furyl)acryloyl-l-tryptophan amide. The strong affinities observed may be due to intense interaction of β-(2-furyl)acryloyl group with a secondary binding site of the enzyme. This interaction led to a k?1k2 value lower than unity, i.e., the rate-limiting process of the acylation was the association, even with the relatively low k2 value of this methyl ester substrate, compared to those proposed for labile p-nitrophenyl esters.  相似文献   

7.
Substitution of the active site zinc ion of carboxypeptidase A by cadmium yields an enzyme inactive towards ordinary peptide substrates. However, a substrate analog (BzGlyNHCH2CSPheOH) containing a thioamide linkage at the scissile position is cleaved to the thioacid. The kinetic parameters and their pH dependencies are kcatKm = 5.04 × 104 min?1M?1, decreasing with either acid or base (PKE1 = 5.64, pKE2 = 9.55), and kcat = 1.02 × 102 min?1, decreasing with acid (pKES = 6.61). The thiopeptide is less efficiently cleaved by native (zinc) carboxypeptidase A. This cadmium-sulfur synergism supports a mechanism wherein the substrate amide is activated by metal ion coordination to its (thio) carbonyl.  相似文献   

8.
A quantitative model for the damping of oscillations of the semiquinone absorption after successive light flashes is presented. It is based on the equilibrium between the states QA?QB and QAQB?. A fit of the model to the experimental results obtained for reaction centers from Rhodopseudomonas sphaeroides gave a value of α = [QA?QB]([QA?QB] + [QAQB?]) = 0.065 ± 0.005 (T = 21°C, pH 8).  相似文献   

9.
(1) H+/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O2, NO?2 or N2O. Under optimal H+-translocation conditions, the ratios H+O, H+N2O, H+NO?2 for reduction to N2 and H+NO?2 for reduction to N2O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO?2 or N2O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. H+N2O, H+NO?2 for reduction to N2 and H+NO?2 for reduction to N2O were ?0.84, ?2.33 and ?1.90, respectively. (3) The H+oxidant ratios, mentioned in item 2, were not altered in the presence of valinomycinK+ and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O2, NO?2 and N2O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO?2, N2O or O2 pass two H+-translocating sites. The H+electron acceptor ratios predicted by this scheme are in good agreement with the experimental values.  相似文献   

10.
The rate of reaction of ferro- and ferricytochrome c (C(II) and C(III)) with ferri- and ferrocyanide and of C(III) with O2? and CO2? was determined in H2O and in 2H2O in the temperature range 5–35 °C. No isotope effect was evident in any of the reductions of C(III); the apparent energy of activation was identical in H2O and 2H2O. An isotope effect with kH2Ok2H2O = 1.25 to 1.85, depending on pH for instance was observed in the oxidation of C(II), in the slow phase of oxidation which involves conformational changes. An interpretation (supported by evidence from previous work) involving water molecules in the close vicinity of the reaction site on the protein is discussed.  相似文献   

11.
The kinetics of bisulfite addition to 5-fluorouracil were studied as a function of increasing concentrations of potential general acids. Values of kobsd[SO3=] measured at 25°C and ionic strength 1.0 M increased linearly and then became invariant with increasing concentrations of either HSO3? or (OHCH2CH2)2N+C(CH2OH)3 HCl (BisTris+HCl). A small kinetic hydrogen-deuterium isotope effect (kHSkDS = 1.10) was observed for the general acid catalysed portion of the addition reaction. The kinetics of bisulfite elimination from 5-fluoro-5,6-dihydrouracil-6-sulfonate were studied in ethanolamine buffers. As previously observed with 1,3-dimethyl-5,6-dihydrouracil-6-sulfonate, this reaction is subject to general base catalysis and exhibits a large kinetic hydrogen-deuterium isotope effect (k2H2Ok2D2O = 3.8). The kinetic results for the addition reaction are consistent with a multistep reaction pathway involving the initial formation of an oxyanion sulfite addition intermediate (II) which subsequently adds a proton and undergoes tautomerization to yield the final 5-fluoro-5,6-dihydrouracil-6-sulfonate product. Thus the elimination of bisulfite from 5-fluoro-5,6-dihydrouracil-6-sulfonate probably proceeds by an ElcB mechanism which involves, at relatively low concentrations of general base, rate determining general base catalyzed proton abstraction from carbon 5 to yield intermediate II followed by the rapid elimination of sulfite to yield 5-fluorouracil. These results may be related to both the enzymatically catalyzed dehalogenation of bromoand iodouracil and the methylation of deoxyuridylate by thymidylate synthetase.  相似文献   

12.
The association constant, KA, for myosin subfragment-1 binding to actin was measured as a function of ionic strength [KCl, LiCl, and tetramethylammonium chloride (TMAC)]and temperature by the method of time-resolved fluorescence depolarization. The following thermodynamic values were obtained from solutions of 0.20 × 10?6m S-1, 1.00 × 10?6m actin in 0.15 m KCl, pH 7.0, at 25 °C: ΔG ° = ?39 ± 1 kJ M?1, ΔH0 = 44 ± 2 kJ M?1 and ΔS0 = 0.28 ± 0.01 kJ M?10K?1. For measurements in KCl (0.05 to 0.60 m), In Ka = ?8.36 (KCl)12. Thus, the binding is endothermic and strongly inhibited by high ionic strength. When KCl was replaced by LiCl or TMAC the ionic effects on the binding were cation specific. The nature of actin-(S-1) binding in the rigor state is discussed in terms of these results.  相似文献   

13.
The magnesium ion-dependent equilibrium of vacant ribosome couples with their subunits
70 S?k?1k150 S+30S
has been studied quantitatively with a novel equilibrium displacement labeling method which is more sensitive and precise than light-scattering. At a concentration of 10?7m, tight couples (ribosomes most active in protein synthesis) dissociate between 1 and 3 mm-Mg2+ at 37 °C with a 50% point at 1.9 mm. The corresponding association constants Ka′ are 5.1 × 105m?1 (1 mm-Mg2+), 3.5 × 107m?1 (2 mm), and 1.2 × 109m?1 (3 mm), about five orders of magnitude higher than the Ka′ value of loose couples studied by Spirin et al. (1971) and Zitomer & Flaks (1972).In this range of Mg2+ concentrations (37 °C, 50 mm-NH4+) the rate constants depend exponentially and in opposite ways on the Mg2+ concentration: k1 = 2.2 × 10?3s?1, k?1 = 7.7 × 104m?1s?1 (2mm-Mg2+); k1 = 1.5 × 10?4s?1, k?1 = 1.7 × 107m?1s?1 (5 mm-Mg2+). Under physiological conditions (Mg2+ ~- 4 mm, ribosome concn ~- 10?7m), the equilibrium strongly favors association and the rate of exchange is slow (t12 ~- 10 min). In the range of dissociation (2 mm-Mg2+), association of subunits proceeds without measurable entropy change and hence ΔGO = ΔHO. The negative enthalpy change of ΔHO = ? 10 kcal suggests that association of subunits involves a shape change.Below a critical Mg2+ concentration (~- 2 mm), the 50 S subunits are converted irreversibly into the b-form responsible for the transition to loose couples. The results are compatible with two classes of binding sites, one class binding Mg2+ non-co-operatively and contributing to the free energy of association by reduction of electrostatic repulsion, and another class probably consisting of hydrogen bonds between components at opposite interfaces whose critical spatial alignment rapidly denatures in the absence of stabilizing magnesium ions.  相似文献   

14.
A quantitative structure-activity relationship has been formulated for 53 alkyl phosphonates [R2OPO(CH3)SR3] inhibiting chymotrypsin: log ki = 1.47MROR2 + 0.34MRSR3 + 1.25σ31 ? 1.06I ? 3.43 log (β·10MROR2 + 1) ? 5.26; log β = ?3.85. In this so-called bilinear model, ki is the bimolecular rate constant (m?1 s?1), β is a disposable parameter evaluated by a computerized iterative procedure, MR is the molar refractivity of a substituent, σ31 is Taft's polar parameter, and I is an indicator variable for substituents containing a sulfonium group. The correlation coefficient for this equation is 0.985. This quantitative structure-activity relationship is compared with those previously formulated for the action of chymotrypsin on acylamino acid ester substrates.  相似文献   

15.
The dephosphorylation of ADP and ATP was characterized as the first-order rate constant in dependence on pH in the absence and presence of Cu2+, and together with Cu2+ and a second ligand. The reaction is strongly accelerated by Cu2+ and passes through pH optima at about 6.2 and 6.5 for the Cu2+ ?ADP and ?ATP systems, respectively (I = 0.1, NaClO4; 50°C). In the presence of 2,2′-bipyridyl (Bipy), ternary complexes are formed with the nucleotides ADP or ATP (NP), Cu(Bipy)(NP), which are very stable towards dephosphorylation over a large pH range. Similar stabilizing effects were observed in ternary complexes formed with imidazole or OH?. These results can easily be rationalized by taking into account that in the binary Cu2+ complexes macrochelates are formed by the interaction between the adenine moiety and the metal ion. This interaction is crucial for obtaining the labile species and hence, in the mixed-ligand complexes, where the macrophelate can not be formed, the phosphates are protected toward hydrolysis. In agreement with these results is the dephosphorylation behavior of Cu(CDP)? and Cu(CTP)2?; they are rather stable. This is in accord with the small coordination tendency of the cytosine moiety.By computing the pH dependence of the distribution of the several species, it is shown that the active species are Cu(ATP)2? and Cu(ADP)? and not the hydroxy complexes, [Cu(ATP)(OH)]26? and [Cu(ADP)(OH)24? as were suggested earlier. With the aid of the initial rate, ν0 = d[PO43?]dt, the rate laws of the ascending side of the pH optima were determined: ν0 = k[Cu(NP)][H+]. The descending side of the pH optima is attributed to the formation of Cu(NP)(OH), where the metal ion interaction with N-7 of the adenine moiety is inhibited.  相似文献   

16.
Systematic heat of dilution studies of the self-association of flavin mononucleotide (FMN) have been conducted as a function of ionic strength (0.05 – 2.0 m) and pH (5–9) in aqueous solution. The data are adequately described by the expression QT = ΔH ? (ΔHK)12 (QTcT)12 for an isodesmic self-association. QT is the molar heat of dilution, ΔH and K are the derived enthalpy and equilibrium constants for the process FMN + (FMN)i?1 ? (FMN)i, and cT is the concentration of FMN expressed in monomer units. Typical values derived for the various thermodynamic parameters at 25 °C are ΔG = ?3.56 kcal mol?1, ΔH = ?3.72 kcal mol?1, and ΔS = ?0.54 cal (mol · deg)?1. These data, plus nuclear magnetic resonance evidence (Yagi, K., Ohishi, N., Takai, A., Kawano, K., and Kyogoku, Y., 1976, Biochemistry15, 2877–2880) argue in favor of an open-ended association of flavin molecules. The signs of the various thermodynamic parameters suggest that both hydrophobic and surface energy forces contribute significantly to the association, while the lack of any significant ionic strength dependence indicates the lack of any ionic centers in the association.  相似文献   

17.
Isolation and characterization of isocitrate lyase of castor endosperm   总被引:1,自引:0,他引:1  
Isocitrate lyase (threo-DS-isocitrate glyoxylate-lyase, EC 4.1.3.1) has been purified to homogeneity from castor endosperm. The enzyme is a tetrameric protein (molecular weight about 140,000; gel filtration) made up of apparently identical monomers (subunit molecular weight about 35,000; gel electrophoresis in the presence of sodium dodecyl sulfate). Thermal inactivation of purified enzyme at 40 and 45 °C shows a fast and a slow phase, each accounting for half of the intitial activity, consistent with the equation: At = A02 · e?k1t + A02 · e?k2t, where A0 and At are activities at time zero at t, and k1 and k2 are first-order rate constants for the fast and slow phases, respectively. The enzyme shows optimum activity at pH 7.2–7.3. Effect of [S]on enzyme activity at different pH values (6.0–7.5) suggests that the proton behaves formally as an “uncompetitive inhibitor.” A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.9. Successive dialysis against EDTA and phosphate buffer, pH 7.0, at 0 °C gives an enzymatically inactive protein. This protein shows kinetics of thermal inactivation identical to the untreated (native) enzyme. Full activity is restored on adding Mg2+ (5.0 mm) to a solution of this protein. Addition of Ba2+ or Mn2+ brings about partial recovery. Other metal ions are not effective.  相似文献   

18.
Acid dissociation constants of aqueous cyclohexaamylose (6-Cy) and cycloheptaamylose (7-Cy) have been determined at 10–47 and 25–55°C, respectively, by pH potentiometry. Standard enthalpies and entropies of dissociation derived from the temperature dependences of these pKa's are ΔH0 = 8.4 ± 0.3 kcal mol?1, ΔS0 = ?28. ± 1 cal mol?10K?1 for 6-Cy and ΔH0 = 10.0 ± 0.1 kcal mol?1, ΔS0 = ?22.4 ±0.3 cal mol?10K?1 for 7-Cy. Intrinsic 13C nmr resonance displacements of anionic 6- and 7-Cy were measured at 30°C in 5% D2O (vv). These results indicate that the dissociation of 6- and 7-Cy involves both C2 and C3 20-hydroxyl groups. The thermodynamic and nmr parameters are discussed in terms of interglucosyl hydrogen bonding.  相似文献   

19.
The dependence of the mitochondrial respiratory rate on the reduction of cytochrome c has been measured as a function of the exogenous [ATP][ADP][Pi] ratio and pH. The respiratory rate at [ADP][ADP][Pi] values of less than 10-1m-1 is proportional to the reduction of cytochrome c and independent of pH from pH 6.5 to pH 8.O. The maximal turnover number (at 100% reduction) for cytochrome c is approximately 70 s?1. As the [ATP][ADP][Pi] ratio is increased from 10?1m?1 to 104m?1, the respiration at any given level of reduction of cytochrome c is progressively inhibited. Greater inhibition is observed at more oxidized levels of cytochorme c with respiratory control values for oxidation of reduced cytochrome c exceeding 10. The behavior of mitochondrial respiratory control is shown to be quantitatively consistent with a proposed mechanism in which the regulation occurs in the reaction of oxygen with cytochrome oxidase. A steady-state rate expression is derived which fits the mitochondrial respiratory rate dependence on (i) the extramitochondrial [ATP][ADP][Pi] ratio; (ii) the level of reduction of cytochrome c (or the intramitochondrial [NAD+][NADH]) at different [ATP][ADP][Pi] values; (iii) the pH of the suspending medium. This rate expression appears to correctly predict the relationships of the cytoplasmic [ATP][ADP][Pi] ratio, the mitochondrial [NAD+][NADH] ratio, and the mitochondrial respiratory rate in intact cells as well as suspensions of isolated mitochondria.  相似文献   

20.
Joël Lunardi  Pierre V. Vignais 《BBA》1982,682(1):124-134
(1) N-4-Azido-2-nitrophenyl-γ-[3H]aminobutyryl-AdoPP[NH]P(NAP4-AdoPP[NH]P) a photoactivable derivative of 5-adenylyl imidodiphosphate (AdoPP[NH]P), was synthesized. (2) Binding of 3H]NAP4-AdoPP[NH]P to soluble ATPase from beef heart mitochrondria (F1) was studied in the absence of photoirradiation, and compared to that of [3H]AdoPP[NH]P. The photoactivable derivative of AdoPP[NH]P was found to bind to F1 with high affinity, like AdoPP[NH]P. Once [3H]NAP4-AdoPP[NH]P had bound to F1 in the dark, it could be released by AdoPP[NH]P, ADP and ATP, but not at all by NAP4 or AMP. Furthermore, preincubation of F1 with unlabeled AdoPP[NH]P, ADP, or ATP prevented the covalent labeling of the enzyme by [3H]NAP4-AdoPP[NH]P upon photoirradiation. (3) Photoirradiation of F1 by [3H]NAP4-AdoPP[NH]P resulted in covalent photolabeling and concomitant inactivation of the enzyme. Full inactivation corresponded to the binding of about 2 mol [3H]NAP4-AdoPP[NH]Pmol F1. Photolabeling by NAP4-AdoPP[NH]P was much more efficient in the presence than in the absence of MgCl2. (4) Bound [3H]NAP4-AdoPP[NH]P was localized on the α- and β-subunits of F1. At low concentrations (less than 10 μM), bound [3H]NAP4-AdoPP[NH]P was predominantly localized on the α-subunit; at concentrations equal to, or greater than 75 μM, both α- and β-subunits were equally labeled. (5) The extent of inactivation was independent of the nature of the photolabeled subunit (α or β), suggesting that each of the two subunits, α and β, is required for the activity of F1. (6) The covalently photolabeled F1 was able to form a complex with aurovertin, as does native F1. The ADP-induced fluorescence enhancement was more severely inhibited than the fluorescence quenching caused by ATP. The percentage of inactivation of F1 was virtually the same as the percentage of inhibition of the ATP-induced fluorescence quenching, suggesting that fluorescence quenching is related to the binding of ATP to the catalytic site of F1.  相似文献   

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