DNA structural polymorphism modulates the kinetics of superhelical DNA cleavage by BamHI restriction endonuclease

A compartmental model developed by Hensley (Hensley, P., Nardone, G., Chirikjian, J.G., and Wastney, M. E., (1990) J. Biol. Chem. 265, 15300-15307) for analysis of the time courses of the cleavage of superhelical DNA substrates by the restriction endonuclease, BamHI, has been used to quantify the effects of changes in temperature, ionic strength, superhelical density, and the DNA substrate on the binding and strand cleavage processes. Studies reported here indicate that changes in topology may be introduced into the DNA substrate solely as a result of the plasmid preparation process and in the absence of covalent bond cleavage and ligation. These changes in topology have qualitatively different effects on the kinetics than those promoted by changes in the superhelical density. The former are removed by briefly warming the DNA prior to assay, suggesting that they are only kinetically stable, while the latter changes are not affected by heating. Increasing the [NaCl] from 0.01 M to 0.1 M increases the overall rate of plasmid cleavage by increasing both the rates of cleavage and enzyme DNA association. To describe the decrease in the overall cleavage rate observed in 0.15 M NaCl, an ionic strength-dependent rate-determining structural transition in the DNA substrate was incorporated into the model. The largest changes in the rate of the cleavage process resulted from changes in the DNA substrate. For the SV40 substrate compared to pBR322, the rate constants describing the two association processes and the first bond cleavage event were increased 6- to 7-fold. The rate of the second bond cleavage process was not affected. These changes may be due to differences in the flanking sequences.     

Catalytic properties of the HhaII restriction endonuclease

The catalytic properties of the HhaII restriction endonuclease were studied using plasmid pSK11 DNA containing a single 5'-G-A-N-T-C HhaII cleavage site as substrate. Reactions were followed by two methods: 1) gel electrophoretic analysis of nicked circular and linear DNA products, or 2) release of 32P-labeled inorganic phosphate from specifically labeled HhaII sites in a reaction coupled with bacterial alkaline phosphatase. The enzyme is optimally active at 37 degrees C in 10 mM Tris-HCl (pH 9.1) and 4-10 mM MgCl2 without added NaCl. Activity is stabilized by the presence of 2-mercaptoethanol and 0.2% Triton X-100 or 50 microgram/ml bovine serum albumin. At enzyme concentrations below 10 nM and using pSK11 as substrate, initial kinetic rates were dependent on the order of mixing of reactants. A lag of 3-4 min was observed if enzyme or substrate was added last. Preincubation of substrate and enzyme followed by initiation of the reaction with MgCl2 or preincubation of the enzyme with nonspecific DNA followed by initiation with substrate eliminated or reduced the lag, respectively, and speeded up the reactions. Under a wide range of reaction conditions, nicked pSK11 DNA accumulated early, while linear molecules appeared later, suggesting that HhaII cleaves one strand at a time in separate binding events. The apparent Km for covalently closed pSK11 DNA molecules was approximately 17 nM, and the turnover number for the conversion of covalent to nicked sites was 1.1 single strand scissions/min. Pre-steady state kinetic analysis indicated that cleavage of the first phosphodiester bond in a site is first order with a rate constant of about 0.8 min-1, while cleavage of the second phosphodiester bond is first order with a rate constant of about 0.2 min-1.     

Photophysics of the fluorescent Ca2+ indicator Fura-2.

The photophysics of the complex forming reaction of Ca2+ and Fura-2 are investigated using steady-state and time-resolved fluorescence measurements. The fluorescence decay traces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution with BAPTA as Ca2+ buffer: k01 = 1.2 x 10(9)s-1, k21 = 1.0 x 10(11) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 2.2 x 10(7) s-1, and with EGTA as Ca2+ buffer: k01 = 1.4 x 10(9) s-1, k21 = 5.0 x 10(10) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 3.2 x 10(7) s-1. k01 and k02 denote the respective deactivation rate constants of the Ca2+ free and bound forms of Fura-2 in the excited state. k21 represents the second-order rate constant of binding of Ca2+ and Fura-2 in the excited state, whereas k12 is the first-order rate constant of dissociation of the excited Ca2+:Fura-2 complex. The ionic strength of the solution was shown not to influence the recovered values of the rate constants. From the estimated values of k12 and k21, the dissociation constant K*d in the excited state was calculated. It was found that in EGTA Ca2+ buffer pK*d (3.2) is smaller than pKd (6.9) and that there is negligible interference of the excited-state reaction with the determination of Kd and [Ca2+] from fluorimetric titration curves. Hence, Fura-2 can be safely used as an Ca2+ indicator. From the obtained fluorescence decay parameters and the steady-state excitation spectra, the species-associated excitation spectra of the Ca2+ free and bound forms of Fura-2 were calculated at intermediate Ca2+ concentrations.     

Purification and properties of the restriction endonuclease BglII from Bacillus globigii

The restriction endonuclease BglII from Bacillus globigii has been purified to homogeneity. The enzyme is a dimer of two subunits of Mr = 27000. The reaction mechanism does not involve the accumulation of a DNA intermediate nicked in one strand and the enzyme is not affected by superhelical twists in the substrate DNA, indicating that DNA binding does not involve either winding or unwinding of the double helix. Antibodies were prepared against BglII. These antibodies did not cross react with any other restriction endonucleases tested, including other enzymes from B. globigii or from closely related strains. It is thus unlikely that type II restriction enzymes represent a closely related group of proteins.     

von Willebrand factor is a cofactor for thrombin-catalyzed cleavage of the factor VIII light chain

The proteolytic activation of highly purified, heterodimeric porcine factor VIII and factor VIII-von Willebrand factor complex by thrombin was compared at I 0.17, pH 7.0, 22 degrees C. During the activation of factor VIII, heavy-chain cleavage is necessary to activate the procoagulant function, whereas light-chain cleavage is required to dissociate factor VIII from von Willebrand factor. The kinetics of activation of free factor VIII and factor VIII-von Willebrand factor complex were identical. The steady-state kinetics of thrombin-catalyzed heavy-chain cleavages and light-chain cleavage of factor VIII either free or in complex with von Willebrand factor were studied using sodium dodecyl sulfate-polyacrylamide gel radioelectrophoresis and scanning densitometry of fragments derived from 125I-labeled factor VIII. Association of factor VIII with von Willebrand factor resulted in an 8-fold increase in the catalytic efficiency (kcat/Km) of light-chain cleavage (from 7 x 10(6) to 54 x 10(6) M-1 s-1). The catalytic efficiencies of heavy-chain cleavage at position 372 (approximately 6 x 10(6) M-1 s-1) and position 740 (approximately 100 x 10(6) M-1 s-1) were not affected by von Willebrand factor. We conclude that von Willebrand factor promotes cleavage of the factor VIII light chain by thrombin which is followed by rapid dissociation of the complex, so that the rate-limiting step becomes heavy-chain cleavage at position 372. This accounts for the observation that von Willebrand factor has no effect on the kinetics of activation of factor VIII by thrombin.     

Thermodynamics and kinetics of the cleavage of DNA catalyzed by bleomycin A5.

Microcalorimetry and UV-vis spectroscopy were used to conduct thermodynamic and kinetic investigations of the scission of calf thymus DNA catalyzed by bleomycin A5 (BLM-A5) in the presence of ferrous ion and oxygen. The molar reaction enthalpy for the cleavage, the Michaelis-Menten constant for calf thymus DNA and the turnover number of BLM-A5 were calculated by a novel thermokinetic method for an enzyme-catalyzed reaction to be -577 +/- 19 kJ.mol-1, 20.4 +/- 3.8 microm and 2.28 +/- 0.49 x 10-2 s-1, respectively, at 37.0 degrees C. This DNA cleavage was a largely exothermic reaction. The catalytic efficiency of BLM-A5 is of the same order of magnitude as that of lysozyme but several orders of magnitude lower than those of TaqI restriction endonuclease, NaeI endonuclease and BamHI endonuclease. By comparing the molar enthalpy change for the cleavage of calf thymus DNA induced by BLM-A5 with those for the scission of calf thymus DNA mediated by adriamycin and by (1,10-phenanthroline)-copper, it was found that BLM-A5 possessed the highest DNA cleavage efficiency among these DNA-damaging agents. These results suggest that BLM-A5 is not as efficient as a DNA-cleaving enzyme although the cleavage of DNA by BLM-A5 follows Michaelis-Menten kinetics. Binding of BLM-A5 to calf thymus DNA is driven by a favorable entropy increase with a less favorable enthalpy decrease, in line with a partial intercalation mode involved in BLM-catalyzed breakage of DNA.     

The kinetic mechanism of beef kidney D-aspartate oxidase

The mechanism of action of the flavoprotein D-aspartate oxidase (EC 1.4.3.1) has been investigated by steady-state and stopped flow kinetic studies using D-aspartate and O2 as substrates in 50 mM KPi, 0.3 mM EDTA, pH 7.4, 4 degrees C. Steady-state results indicate that a ternary complex containing enzyme, O2, and substrate (or product) is an obligatory intermediate in catalysis. The kinetic parameters are turnover number = 11.1 s-1, Km(D-Asp) = 2.2 x 10(-3) M, Km(O2) = 1.7 x 10(-4) M. Rapid reaction studies show that 1) the reductive half reaction is essentially irreversible with a maximum rate of reduction of 180 s-1; 2) the free reduced enzyme cannot be the species which is reoxidized during turnover since its reoxidation by oxygen (second order rate constant equal to 5.3 x 10(2) M-1 s-1) is too slow to be of relevance in catalysis; 3) reduced enzyme can bind a ligand rapidly and be reoxidized as a complex at a rate faster than that observed for the free reduced enzyme; 4) the rate of reoxidation of reduced enzyme by oxygen during turnover is dependent on both O2 and D-aspartate concentrations (second order rate constant of reaction between O2 and reduced enzyme-substrate complex equal to 6.2 x 10(4) M-1 s-1); and 5) the rate-limiting step in catalysis occurs after reoxidation of the enzyme and before its reduction in the following turnover. A mechanism involving reduction of enzyme by substrate, dissociation of product from reduced enzyme, binding of a second molecule of substrate to the reduced enzyme, and reoxidation of the reduced enzyme-substrate complex is proposed for the enzyme-catalyzed oxidation of D-aspartate.     

Interaction of RNA polymerase with promoters from bacteriophage fd.

Replicative form DNA of bacteriophage fd, which had been fragmented with the restriction endonuclease II from Hemophilus parainfluenzae (endo R- HpaII), was reacted with Escherichia coli RNA polymerase; the resulting stable preinitiation complexes were analysed using the filter binding assay followed by gel electrophoresis. At 120mM KCL the first-order rate constants for complex decay were determined to be 10(-2)-10(-6)s-1. The second-order rate constants for complex formation were found to be about 10(6) -10(7) M-1 s-1. From these values association constants for the individual promoters were calculated to be 2 x 10(-8) -2 x 10(-11) M-1. The rate of formation and the stability of promoter complexes was enhanced in superhelical DNA. No evidence was found for stable promoter-specific closed complexes consisting of enzyme and helical DNA. This and the kinetic data suggest that the unwinding of base pairs is already important early in promoter selection, and not only for the formation of the final open complex. The initiation of RNA synthesis form the preinitiation complex was faster than complex dissociation and complex formation for all promoters. Consequently, the initiation efficiency of a promoter is determined by the rate of complex formation, and not by its 'affinity' for the enzyme. No correlation was found between the relative order of the fd promoters for the binding and the dissociation reaction. This is explained by different structural determinants, for the two reactions, which are located in different parts of the promoter DNA.     

An endonuclease activity of venom phosphodiesterase specific for single-stranded and superhelical DNA.

A homogeneous preparation of venom phosphodiesterase from Crotalus adamanteus possesses an intrinsic endonuclease activity, specific for superhelical (form I) and single-stranded DNA. The phosphodiesterase degrades single-stranded T7 DNA by endonucleolytic cleavages. Duplex T7 DNA is hydrolyzed by the liberation of acid-soluble products simultaneously from the 3' and 5' termini but without demonstrable internal scissions in duplex regions. Since venom phosphodiesterase is known to hydrolyze oligonucleotides stepwise from the 3' termini, the cleavage at the 5' end of duplex T7 DNA is ascribed to an endonuclease activity. Form I PM2 DNA is nicked to yield first relaxed circles and then linear DNA which is subsequently hydrolyzed only from the chain termini. The linear duplex DNA intermediates consist of a discrete series of fragments (11 are usually resolved on agarose gels) with initial molecular weights ranging from 6.3 x 10(6) (the intact PM2 DNA size) to approximately 1 x 10(6). The cleavage of the form I molecule must, therefore, occur at a limited number of unique sites. The enzyme also cleaves nonsuperhelical, covalently closed circular PM2 DNA but at a 10(4) times slower rate. Both the endonuclease activity on form I DNA and the known exonuclease activity co-migrate on polyacrtkanude gels, are optimally active at pH 9, are stimulated by small concentrations of Mg2+, and are similarly inactivated by heat, reducing agents, and EDTA.     

Relaxed circular SV40 DNA as cleavage intermediate of two restriction endonucleases.

We have determined the mode of cleavage of superhelical SV40 DNA (Form I) by restriction endonucleases EcoRI and HpaII at 37 degrees C. By analysis with agarose gel electrophoresis and direct examination with dark field electron microscopy, we found that a large amount of the single-nicked circular DNA (Form II) was produced before the linear SV40 DNA (Form III) appeared. Thus, both restriction enzymes cleave only one strand of the superhelical DNA first. The second cleavage on the complementary strand occurred after a lag period. The first order rate constant for the second cleavage by EcoRI endonuclease was determined and a kinetic reaction scheme for both enzymes is proposed.     

Transient cleavage kinetics of the Eco RI restriction endonuclease measured in a pulsed quench-flow apparatus: enzyme concentration-dependent activity change.

We report measurements of the cleavage rate of pBR 322 plasmid DNA by the restriction endonuclease Eco RI as a function of enzyme and DNA concentration. The reaction, which at high excess of enzyme over DNA occurs between 0.2 and 5 seconds, was studied by the means of a microprocessor controlled pulsed quench-flow apparatus. Enzyme concentrations were between 1 and 100 nM with DNA concentrations being 3 to 6 nM (specific Eco RI sites). The catalytic constants for cleavage of the first and second phosphodiester bonds as measured at high enzyme concentration both have the same value of 0.35 sec-1 and 21 degrees C. At enzyme concentrations comparable to or less than DNA concentration, the rate of the first cleavage is proportional to enzyme concentration, while the second step is independent of concentration. At approx. 10 nM Eco RI endonuclease concentration, a rate increase shows up in both the first and the second cleavage. We suggest that this increase is due to the tetramerization reported by Modrich & Zabel1, which occurs in this concentration range.     

Binding of ellipticine base and ellipticinium cation to calf-thymus DNA. A thermodynamic and kinetic study

The acid-basic properties of ellipticine have been re-estimated. The apparent pK of protonation at 3 microM drug concentration is 7.4 +/- 0.1. The ellipticine free base (at pH 9, I = 25 mM) intercalates into calf-thymus DNA with an affinity constant of 3.3 +/- 0.2 X 10(5) M-1, and a number of binding sites per phosphate of 0.23. The ellipticinium cation (pH 5, I = 25 mM) binds also to DNA with a constant of 8.3 +/- 0.2 x 10(5) M-1 and at a number of binding sites (n = 0.19). It is postulated that the binding of the drug to DNA at pH 9 is driven by hydrophobic and/or dipolar effects. Even at pH 5, where ellipticine exists as a cation, it is thought that the hydrophobic interaction is the main contribution to binding. The neutral and cationic forms share common binding within DNA sites but yield to structurally different complexes. The free base has 0.04 additional specific binding sites per phosphate. As determined from temperature-jump experiments, the second-order rate constant of the binding of the free base (pH 9) is 3.4 x 10(7) M-1 s-1 and the residence time of the base within the DNA is 8 ms. The rate constant for the binding of the ellipticinium cation is 9.8 x 10(7) M-1 s-1 when it is assumed that drug attachment occurs via a pathway in which the formation of an intermediate ionic complex is not involved (competitive pathway).     

Kinetic parameters of superhelical DNA cleavage by endonuclease S1

Major kinetic parameters of endonuclease S1 were determined on superhelical bacteriophage PM2 DNA and on relaxed nicked circular PM2 DNA. At 37 degrees and 0,25 M NaCl, the Michaelis constants were respectively 1.7 . 10(-8) M and 1 . 10(-9) M, and catalytic constants were respectively 0.36 sec-1 and 1.2 . 10(-2) sec-1. The inhibition of the enzyme reaction by its product was detected.     

Role of the 2-amino group of deoxyguanosine in sequence recognition by EcoRI restriction and modification enzymes.

The dG residues within the EcoRI recognition sequence of ColE1 DNA have been selectively replaced with dI. Methylation of the altered sequence by the EcoRI modification enzyme is extremely slow as compared with methyl transfer to the natural recognition site. Since the affinity of the modification enzyme for the dI-containing sequence is considerably less than that for the natural sequence, we have concluded that the 2-amino group of dG has an important role in DNA site recognition by this enzyme. In contrast, the altered site is subject to cleavage by EcoRI endonuclease at rates essentially identical with those observed with the natural sequence. These results strongly suggest that the two enzymes utilize different contacts within the EcoRI site and are consisted with our conclusion (Rubin, R. A., and Modrich, P. (1977) J. Biol. Chem. 252, 7265-7272) that the two proteins interact with their common recognition sequence in different ways.     

An endonucleolytic activity of nuclease TT1 specific for superhelical DNA.

Highly purified nuclease TT1 from T. thermophilus HB8 acts on a linear single- and double-stranded DNA as an exonuclease and produces 5'-mononucleotides either from the 5'- or 3'-terminus. It was found that the enzyme also possesses an endonuclease activity specific for superhelical (form I) and single-stranded circular DNA. Form I of various kinds of DNA (phi X174, PM2, Co1E1 and RF 1010 etc.) is nicked to yield first relaxed circles (form II) and then nicked at the opposite site to yield unit length linear DNA (form III), which is subsequently hydrolyzed from the 5'- or 3'-terminus. A single cleavage of the form I of phi X174 DNA seemed to occur at a limited number of unique sites. Both endonuclease and the known exonuclease activities co-migrate on polyacrylmide gels, show the same pH and temperature optima, are stimulated by Mg2+ and are inactivated by EDTA similarly.     

Palmitate binding to and efflux kinetics from human erythrocyte ghost

At 0 degrees C, pH 7.3, palmitate (PA) binds to human erythrocyte ghosts suspended in 0.2% bovine serum albumin (BSA) solution with molar ratios of PA to BSA, v, between 0.2 and 1.3. The binding depends on the water phase PA concentration, measured in equilibrium experiments, using BSA-filled ghosts as semipermeable bags. The saturable binding has a capacity of 19.4 +/- 7.5 nmol g-1 packed ghosts (7.2 x 10(9) cells) and Kd = 13.5 +/- 5 nM. PA exchange efflux kinetics to 0.2% BSA is recorded from ghosts without and with 0.2% BSA with a resolution time of about 1 s. Data are analyzed in terms of compartmental models. Using BSA-free ghosts the kinetics is essentially monoexponential. The rate constant is 0.0287 +/- 0.0022 s-1. Using ghosts with BSA, the kinetics is biexponential with widely different rate constants. Extrapolated zero-time values reflect, according to additional investigations, 'instantaneous' release of PA from the outer surface of the ghosts. Analyses of the biexponential curve up to about 55% tracer efflux assign unequivocally values to three model parameters. (1) k1, the dissociation rate constant of the PA-BSA complex is (1.47 +/- 0.03) x 10(-3) s-1 and (2.56 +/- 0.08) x 10(-3) s-1 and (4.08 +/- 0.13) x 10(-3) s-1 at v = 0.2, 0.6 and 1.4, respectively. (2) k3*, the overall rate constant of PA transport from the inside of the ghost membrane to the medium is 0.0269 +/- 0.0020 s-1 independent of v. (3) Qkin, the ratio of PA on the inside of the membrane to PA on BSA within the ghosts is v dependent and smaller than a corresponding ratio Qeq measured in equilibrium by a value corresponding to PA on the outer surface. This fraction is released with a rate constant, k5, which is of the order of 1 s-1. The data suggest a maximum PA transport capacity, Jmax, of 2 pmol min-1 cm-2, 0 degrees C, pH 7.3.     

Stopped-flow kinetic study of the peroxidase reactions of mangano-microperoxidase-8

We have investigated the kinetics for the peroxidase-type reaction of mangano microperoxidase 8 (Mn(III)-MP8) by the time-resolved and single-wavelength stopped-flow technique. The formation of intermediate and its subsequent reaction with substrates were studied separately. Oxidation of Mn(III)-MP8 by H2O2 at pH 10.7 yields an intermediate (1) with a rate constant of 2.9 x10(4) M-1 s-1. The formation of 1 exhibits no deuterium solvent isotope effect, favoring the homolytic cleavage of the Mn(III)-MP8 bound hydroperoxide. The rate for the formation of 1 increases sharply as the pH increases and no other intermediate was detected in the entire pH range. Addition of substrate to 1 leads to the regeneration of Mn(III)-MP8. Monitoring the conversion of 1 to Mn(III)-MP8 allows the determination of the substrate reactivity. The substrate reactivity varies by more than two orders of magnitude ranging from 1.04 x 10(6) M-1 s-1 for ascorbic acid to 4.61 x 10(3) M-1s-1 for aniline. It is linearly correlated with the reduction potential for most of the substrates studied, with the easier oxidized species showing greater reactivity. The substrate reactivity drops rapidly as the pH increases. The substrate reactivity at pH 10.7 for the Mn(III)-MP8 system is smaller than that of the corresponding Fe(III)-MP8 system by 2- to 25-fold, depending on the substrate used.     

Kinetic analysis of oligodeoxyribonucleotide-directed triple-helix formation on DNA

Pyrimidine oligonucleotides recognize extended purine sequences in the major groove of double-helical DNA by triple-helix formation. The resulting local triple helices are relatively stable and can block DNA recognition by sequence-specific DNA binding proteins such as restriction endonucleases. Association and dissociation kinetics for the oligodeoxyribonucleotide 5'-CTCTTTCCTCTCTTTTTCCCC (bold C's indicate 5-methylcytosine residues) are now measured with a restriction endonuclease protection assay. When oligonucleotides are present in greater than 10-fold excess over the DNA target site, the binding reaction kinetics are pseudo first order in oligonucleotide concentration. Under our standard conditions (37 degrees C, 25 mM Tris-acetate, pH 6.8, 70 mM sodium chloride, 20 mM magnesium chloride, 0.4 mM spermine tetrahydrochloride, 10 mM beta-mercaptoethanol, 0.1 mg/mL bovine serum albumin) the value of the observed pseudo-first-order association rate constant, k2obs, is 1.8 x 10(3) +/- 1.9 x 10(2) L.(mol of oligomer-1.s-1. Measurement of the dissociation rate constant yields an equilibrium dissociation constant of approximately 10 nM. Increasing sodium ion concentration slightly decreased the association rate, substantially increased the dissociation rate, and thereby reduced the equilibrium binding constant. This effect was reversible by increasing multivalent cation concentration, confirming the significant role of multivalent cations in oligonucleotide-directed triple-helix formation under these conditions. Finally, a small reduction in association rate, a large increase in dissociation rate, and a resulting reduction in the equilibrium binding constant were observed upon increasing the pH between 6.8 and 7.2.     

The relation of acyl transfer to the overall reaction of thiolase I from porcine heart

Thiolase I (long chain 3-ketoacyl-CoA-specific) from porcine heart has been characterized kinetically. In the direction of acetoacetyl-CoA cleavage, a variety of thiols including CoASH show the same Vmax at saturating concentrations of acetoacetyl-CoA. At a constant overall velocity of acetoacetyl-CoA disappearance, one of the two acetyl groups from acetoacetyl-CoA will partition between CoASH and 2-mercaptoethanol at increasing 2-mercaptoethanol concentrations. These observations suggest rate-determining formation of an acetyl enzyme intermediate in the direction of acetoacetyl-CoA cleavage. In the direction of acetoacetyl-CoA formation from two molecules of acetyl-CoA, the Vmax of acetoacetyl-CoA formation is identical with the Vmax for an acetyl-CoA in equilibrium CoA isotope exchange reaction and the Vmax for an enzyme-catalyzed acetyl transfer reaction between acetyl-CoA and 2-mercaptoethanol. This suggests that in the direction of acetoacetyl-CoA synthesis, the acetyl transfer half-reaction is rate-limiting. The acetyl intermediate has been isolated and characterized. The equilibrium constant for acetyl enzyme formation from acetyl-CoA and free enzyme is 1 +/- 0.5 X 10(-2). The rate constant for spontaneous hydrolysis of the acetyl enzyme (2.6 X 10(-4) s-1) is a factor of 400 faster than the rate constant for acetyl-CoA hydrolysis under comparable conditions. The acetyl enzyme is thermodynamically and kinetically destabilized compared to acetyl-CoA.     

Photophysics of the fluorescent K+ indicator PBFI.

The fluorescent indicator PBFI is widely used for the determination of intracellular concentrations of K+. To investigate the binding reaction of K+ to PBFI in the ground and excited states, steady-state and time-resolved measurements were performed. The fluorescence decay surface was analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution at pH 7.2: k01 = 1.1 x 10(9) s-1, k21 = 2.7 x 10(8) M-1s-1, k02 = 1.8 x 10(9) s-1, and k12 = 1.4 x 10(9) s-1. k01 and k02 denote the respective deactivation rate constants of the K+ free and bound forms of PBFI in the excited state. k21 represents the second-order rate constant of binding of K+ to the indicator in the excited state whereas k12 is the first-order rate constant of dissociation of the excited K(+)-PBFI complex. From the estimated values of k12 and k21, the dissociation constant Kd* in the excited state was calculated. It was found that pKd* (-0.7) is smaller than pKd (2.2). The effect of the excited-state reaction can be neglected in the determination of Kd and/or the K+ concentration. Therefore, intracellular K+ concentrations can be accurately determined from fluorimetric measurements by using PBFI as K+ indicator.