Rate-determining folding and association reactions on the reconstitution pathway of porcine skeletal muscle lactic dehydrogenase after denaturation by guanidine hydrochloride

Reactivation of tetrameric porcine skeletal muscle lactic dehydrogenase after dissociation and extensive unfolding of the monomers by 6 M guanidine hydrochloride (Gdn . HCl) is characterized by sigmoidal kinetics, indicating a complex mechanism involving rate-limiting folding and association steps. For analysis of the association reactions, chemical cross-linking with glutaraldehyde may be used [Hermann, R., Jaenicke, R., & Rudolph, R. (1981) Biochemistry 20, 2195-2201]. The data clearly show that the formation of a dimeric intermediate is determined by a first-order folding reaction of the monomers with k1 = (8.0 +/- 0.1) x 10(-4) s-1. The rate constant of the association of dimers to tetramers which represents the second rate-limiting step on the pathway of reconstitution after guanidine denaturation, was then determined by reactivation and cross-linking experiments after dissociation in 0.1 M H3PO4 containing 1 M Na2SO4. The rate constant for the dimer association (which is the only rate-limiting step after acid dissociation) was k2 = (3.0 +/- 0.5) x 10(4) M-1 s-1. On the basis of the given two rate constants, the complete reassociation pattern of porcine lactic dehydrogenase after dissociation and denaturation in 6 M Gdn . HCl can be described by the kinetic model (formula: see text).     

Static and kinetic studies on carp muscle parvalbumins

Fluorescence titration and fluorescence stopped-flow studies were performed on carp muscle parvalbumin components 1, 2, 3, and 5 (the latter three components were modified with a SH-directed fluorescent reagent, dansyl-L-cysteine). Apparent binding constants (Kapp) of Ca2+ to these components decrease in the order of component 2 (Kapp = 2.8 +/- 0.9 X 10(8) M-1) greater than component 1 (Kapp = 1.25 +/- 0.25 X 10(8) M-1) greater than component 3 = component 5 (Kapp = 4.0 +/- 0.5 X 10(7) M-1) in 30 mM KCl, 50 mM Na-cacodylate-HCl, pH 7.0 at 20 degrees C. The rate constant of the conformational change of parvalbumin induced by Ca2+ binding or removal decreases in the order of component 2 greater than component 1 greater than component 5 greater than or equal to component 3; that is, component 2 undergoes the fastest conformational change and component 3 the slowest in response to the rapid free Ca2+ concentration ([Ca2+]) change in the protein solution. The fluorescence titration curves and [Ca2+]-dependences of the rate constants are analyzed by a simple two-state model, (partially unfolded state) k1 in equilibrium k2 (folded state). It is shown that the equilibrium constant K = k1/k2 depends on the second power of [Ca2+], the rate constant k1 on the first power of [Ca2+] and k2 on the inverse first power of [Ca2+], respectively.     

Thermodynamics of the reduction of NADP with 2-propanol catalyzed by an NADP-dependent alcohol dehydrogenase

The apparent equilibrium constant of the biochemical reaction, 2-propanol+NADP(ox) = acetone+NADP(red), was determined at I = 0.25 M over a wide range of pH (5.63 to 8.02) and temperature (5 to 40 degrees C). The reaction was catalyzed by an NADP-dependent alcohol dehydrogenase. The results were used to calculate thermodynamic quantities for the chemical (ionic) reference reaction: 2-propanol+NADP(ox)(3-) = acetone+NADP(red)(4-)+H(+). The thermodynamic quantities for this reference reaction are as follows: equilibrium constant K = (5.98+/-0.46) x 10(-10); standard molar Gibbs energy change Delta(r)G(0) = (52.65+/-0.19) kJmol(-1); standard molar enthalpy change Delta(r)H(0) = (38.9+/-0.6) kJmol(-1); and standard molar entropy change Delta(r)S(0) = -(46.1+/-2.2)J K(-1)mol(-1). All of these results pertain to 25 degrees C (298.15 K) and I = 0. The results also lead, in conjunction with tabulated thermodynamic quantities, to the standard electromotive force E(0) = -0.140 V for the reduction of NADP(ox)(3-) to NADP(red)(4-).     

Dye-induced aggregation of single stranded RNA: a mechanistic approach

The binding of proflavine (D) to single stranded poly(A) (P) was investigated at pH 7.0 and 25 degrees C using T-jump, stopped-flow and spectrophotometric methods. Equilibrium measurements show that an external complex PD(I) and an internal complex PD(II) form upon reaction between P and D and that their concentrations depend on the polymer/dye concentration ratio (C(P)/C(D)). For C(P)/C(D)<2.5, cooperative formation of stacks external to polymer strands prevails (PD(I)). Equilibria and T-jump experiments, performed at I=0.1M and analyzed according to the Schwarz theory for cooperative binding, provide the values of site size (g=1), equilibrium constant for the nucleation step (K( *)=(1.4+/-0.6)x10(3)M(-1)), equilibrium constant for the growth step (K=(1.2+/-0.6)x10(5)M(-1)), cooperativity parameter (q=85) and rate constants for the growth step (k(r)=1.2x10(7)M(-1)s(-1), k(d)=1.1 x 10(2)s(-1)). Stopped-flow experiments, performed at low ionic strength (I=0.01 M), indicate that aggregation of stacked poly(A) strands do occur provided that C(P)/C(D)<2.5.     

Assay and purification of the adenosylcobalamin-dependent 2-methyleneglutarate mutase from Clostridium barkeri

A continuous spectrophotometric assay was developed for the adenosylcobalamin-dependent 2-methyleneglutarate mutase from Clostridium barkeri. Thereby the product (R)-3-methylitaconate is converted by the delta-isomerase from the same organism to 2,3-dimethylmaleate which absorbs at 240 nm, much higher than both parent compounds (delta epsilon = 3.7 mM-1.cm-1). In addition a discontinuous assay using the facile formation of 2,3-dimethylmaleic anhydride in aqueous solution at pH 0-1 (delta epsilon = 4.0 mM-1.cm-1 at 256 nm) was established. The mutase and the isomerase were purified together by chromatography on quaternary-amine-Sepharose (Q-Sepharose) and on cyanocobalamin-agarose. The enzymes were separated and obtained in homogenous forms by preparative PAGE in non-denaturing buffer. Both enzymes appear to be homotetramers with subunits of 70 kDa (mutase) and 50 kDa (isomerase). The equilibrium constants for both reactions were determined at I = 0.1 M and 25 degrees C: K1, app = [(R)-3-methylitaconate].[2-methyleneglutarate]-1 = 0.26 +/- 0.04, K2,app = [2,3-dimethylmaleate].[(R)-3-methylitaconate]-1 = 7.40 +/- 0.21.     

Azide binding to yeast cytochrome c peroxidase and horse metmyoglobin: comparative thermodynamic investigation using isothermal titration calorimetry

Yeast cytochrome c peroxidase (CcP) and horse metmyoglobin (Mb) bind HN3 with similar affinities at 25 degrees C. The pH-independent equilibrium association constants for formation of the CcP.HN3 and Mb.HN3 complexes are (1.05 +/- 0.06)x10(5) and (1.6 +/- 0.8)x10(5) M(-1), respectively. However, the thermodynamic parameters for formation of the two complexes are quite different. The DeltaH0 values for formation of CcP.HN3 and Mb.HN3 are -16.4 +/- 0.7 and -9.0 +/- 0.5 kcal/mol, respectively, and the Delta S0 values are -32 +/- 2 and -16 +/- 2 cal/deg mol, respectively. The proton associated with HN3 is retained in both protein complexes at low pH but dissociates with apparent pKA values of 5.5 +/- 0.2 and > or =8.2 for the Mb.HN3 and CcP.HN3 complexes, respectively. CcP and Mb differ significantly in their reactivity toward the azide anion, N3-. CcP binds N3- very weakly, if at all, and only an upper-limit of 18 +/-5 M(-1) for the pH-independent equilibrium association constant for the CcP.N3- complex can be determined. Mb binds N3- with an association constant of (1.8 +/- 0.1)x10(4) M(-1). The ratio of the equilibrium association constants for HN3 and N3- binding provides a discrimination factor between the neutral and charged forms of the ligand. The discrimination factor is greater than 5800 for CcP but only nine for Mb. Protonation of the distal histidines in the two proteins influences binding of HN3. Protonation of His-64 in Mb enhances HN3 binding due to a gating mechanism while protonation of His-52 in CcP decreases the affinity for HN3 due to loss of base-assisted association of the ligand to the heme iron.     

Cobalt and the iron acquisition pathway: competition towards interaction with receptor 1

During iron acquisition by the cell, complete homodimeric transferrin receptor 1 in an unknown state (R1) binds iron-loaded human serum apotransferrin in an unknown state (T) and allows its internalization in the cytoplasm. T also forms complexes with metals other than iron. Are these metals incorporated by the iron acquisition pathway and how can other proteins interact with R1? We report here a four-step mechanism for cobalt(III) transfer from CoNtaCO(3)(2-) to T and analyze the interaction of cobalt-loaded transferrin with R1. The first step in cobalt uptake by T is a fast transfer of Co(3+) and CO(3)(2-) from CoNtaCO(3)(2-) to the metal-binding site in the C-lobe of T: direct rate constant, k(1)=(1.1+/-0.1) x 10(6) M(-1) s(-1); reverse rate constant, k(-1)=(1.9+/-0.6) x 10(6) M(-1) s(-1); and equilibrium constant, K=1.7+/-0.7. This step is followed by a proton-assisted conformational change of the C-lobe: direct rate constant, k(2)=(3+/-0.3) x 10(6) M(-1) s(-1); reverse rate constant, k(-2)=(1.6+/-0.3) x 10(-2) s(-1); and equilibrium constant, K(2a)=5.3+/-1.5 nM. The two final steps are slow changes in the conformation of the protein (0.5 h and 72 h), which allow it to achieve its final thermodynamic state and also to acquire second cobalt. The cobalt-saturated transferrin in an unknown state (TCo(2)) interacts with R1 in two different steps. The first is an ultra-fast interaction of the C-lobe of TCo(2) with the helical domain of R1: direct rate constant, k(3)=(4.4+/-0.6)x10(10) M(-1) s(-1); reverse rate constant, k(-3)=(3.6+/-0.6) x 10(4) s(-1); and dissociation constant, K(1d)=0.82+/-0.25 muM. The second is a very slow interaction of the N-lobe of TCo(2) with the protease-like domain of R1. This increases the stability of the protein-protein adduct by 30-fold with an average overall dissociation constant K(d)=25+/-10 nM. The main trigger in the R1-mediated iron acquisition is the ultra-fast interaction of the metal-loaded C-lobe of T with R1. This step is much faster than endocytosis, which in turn is much faster than the interaction of the N-lobe of T with the protease-like domain. This can explain why other metal-loaded transferrins or a protein such as HFE-with a lower affinity for R1 than iron-saturated transferrin but with, however, similar or higher affinities for the helical domain than the C-lobe-competes with iron-saturated transferrin in an unknown state towards interaction with R1.     

On the dehydration of (R)-lactate in the fermentation of alanine to propionate by Clostridium propionicum

All the enzymes of the pathway of (S)-alanine fermentation to acetate and propionate were detected in cell-free extracts of Clostridium propionicum . Among these (S)-glutamate dehydrogenase (NAD), (R)-lactate dehydrogenase (NAD) and propionate CoA-transferase were purified to apparent homogeneity. Their structures were presumably alpha 6, alpha 2 and alpha 4, respectively. The latter enzyme was specific for short-chain monocarboxylic acids with a pronounced preference for (R)-lactate over the (S)-enantiomer. The key step of the pathway, the dehydration of (R)-lactate required acetyl phosphate and CoASH under anaerobic conditions. It was inhibited by hydroxylamine, arsenate, azide (1 mM each) or by 0.1 mM 2,4-dinitrophenol. Thus it closely resembled the dehydration of (R)-2-hydroxyglutarate in Acidaminococcus fermentans , although an activation was not necessary.     

Structural basis for stereo-specific catalysis in NAD(+)-dependent (R)-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans

NAD(+)-dependent (R)-2-hydroxyglutarate dehydrogenase (HGDH) catalyses the reduction of 2-oxoglutarate to (R)-2-hydroxyglutarate and belongs to the d-2-hydroxyacid NAD(+)-dependent dehydrogenase (d-2-hydroxyacid dehydrogenase) protein family. Its crystal structure was determined by phase combination to 1.98 A resolution. Structure-function relationships obtained by the comparison of HGDH with other members of the d-2-hydroxyacid dehydrogenase family give a chemically satisfying view of the substrate stereoselectivity and catalytic requirements for the hydride transfer reaction. A model for substrate recognition and turnover is discussed. The HGDH active site architecture is structurally optimized to recognize and bind the negatively charged substrate 2-oxoglutarate. The structural position of the side chain of Arg52, and its counterparts in other family members, strongly correlates with substrate specificity towards substitutions at the C3 atom (linear or branched substrates). Arg235 interacts with the substrate's alpha-carboxylate and carbonyl groups, having a dual role in both substrate binding and activation, and the gamma-carboxylate group can dock at an arginine cluster. The proton-relay system built up by Glu264 and His297 permits His297 to act as acid-base catalyst and the 4Re-hydrogen from NADH is transferred as hydride to the carbonyl group Si-face leading to the formation of the correct enantiomer (R)-2-hydroxyglutarate.     

Thermodynamics of the arginine kinase reaction.

The effect of temperature, pH, free [Mg(2+)], and ionic strength on the apparent equilibrium constant of arginine kinase (EC 2.7.3.3) was determined. At equilibrium, the apparent K' was defined as [see text] where each reactant represents the sum of all the ionic and metal complex species. The K' at pH 7.0, 1.0 mM free [Mg(2+)], and 0. 25 M ionic strength was 29.91 +/- 0.59, 33.44 +/- 0.46, 35.44 +/- 0. 71, 39.64 +/- 0.74, and 45.19 +/- 0.65 (n = 8) at 40, 33, 25, 15, and 5 degrees C, respectively. The standard apparent enthalpy (DeltaH degrees') is -8.19 kJ mol(-1), and the corresponding standard apparent entropy of the reaction (DeltaS degrees') is + 2. 2 J K(-1)mol(-1) in the direction of ATP formation at pH 7.0, free [Mg(2+)] =1.0 mM, ionic strength (I) =0.25 M at 25 degrees C. We further show that the magnitude of transformed Gibbs energy (DeltaG degrees ') of -8.89 kJ mol(-1) is mostly comprised of the enthalpy of the reaction, with 7.4% coming from the entropy TDeltaS degrees' term (+0.66 kJ mol(-1)). Our results are discussed in relation to the thermodynamic properties of its evolutionary successor, creatine kinase.     

Intercalation of proflavine and a platinum derivative of proflavine into double-helical Poly(A)

The equilibria and kinetics of the interactions of proflavine (PR) and its platinum-containing derivative [PtCl(tmen)(2)HNC(13)H(7)(NHCH(2)CH(2))(2)](+) (PRPt) with double-stranded poly(A) have been investigated by spectrophotometry and Joule temperature-jump relaxation at ionic strength 0.1 M, 25 degrees C, and pH 5.2. Spectrophotometric measurements indicate that base-dye interactions are prevailing. T-jump experiments with polarized light showed that effects due to field-induced alignment could be neglected. Both of the investigated systems display two relaxation effects. The kinetic features of the reaction are discussed in terms of a two-step series mechanism in which a precursor complex DS(I) is formed in the fast step, which is then converted to a final complex in the slow step. The rate constants of the fast step are k(1) = (2.5 +/- 0.4) x 10(6) M(-1) s(-1), k(-1) = (2.4 +/- 0.1) x 10(3) s(-1) for poly(A)-PR and k(1) = (2.3 +/- 0.1) x 10(6) M(-1) s(-1), k(-1) = (1.6 +/- 0.2) x 10(3) s(-1) for poly(A)-PRPt. The rate constants for the slow step are k(2) = (4.5 +/- 0.5) x 10(2) s(-1), k(-2) = (1.7 +/- 0.1) x 10(2) s(-1) for poly(A)-PR and k(2) = 9.7 +/- 1.2 s(-1), k(-2) = 10.6 +/- 0.2 s(-1) for poly(A)-PRPt. Spectrophotometric measurements yield for the equilibrium constants and site size the values K = (4.5 +/- 0.1) x 10(3) M(-1), n = 1.3 +/- 0.5 for poly(A)-PR and K = (2.9 +/- 0.1) x 10(3) M(-1), n = 2.3 +/- 0.6 for poly(A)-PRPt. The values of k(1) are similar and lower than expected for diffusion-limited reactions. The values of k(-1) are similar as well. It is suggested that the formation of DS(I) involves only the proflavine residues in both systems. In contrast, the values of k(2) and k(-2) in poly(A)-PRPt are much lower than in poly(A)-PR. The results suggest that in the complex DS(II) of poly(A)-PRPt both proflavine and platinum residues are intercalated. In addition, a very slow process was detected and ascribed to the covalent binding of Pt(II) to the adenine.     

Catalase-peroxidase from the cyanobacterium Synechocystis PCC 6803: cloning, overexpression in Escherichia coli, and kinetic characterization.

The Synechocystis PCC 6803 katG gene encodes a dual-functional catalase-peroxidase (EC 1.11.1.7). We have established a system for the high level expression of a fully active recombinant form of this enzyme. Its entire coding DNA was extended using a synthetic oligonucleotide encoding a hexa-histidine tag at the C-terminus and expressed in Escherichia coli [BL21-(DE3)pLysS] using the pET-3a vector. Hemin was added to the culture medium to ensure its proper association with KatG upon induction. The expressed protein was purified to homogeneity by two chromatography steps including a metal chelate affinity and hydrophobic interaction chromatography. The homodimeric acidic protein (pl = 5.4) had a molecular mass of 170 kDa and a Reinheitszahl (A406/A280) of 0.64. The recombinant protein contained high catalase activity (apparent Km = 4.9 +/- 0.25 mM and apparent kcat = 3500 s(-1)) and an appreciable peroxidase activity with o-dianisidine, guaiacol and pyrogallol, but not with NAD(P)H, ferrocytochrome c, ascorbate or glutathione as electron donors. By using both conventional and sequential stopped-flow spectroscopy, formation of compound I with peroxoacetic acid was calculated to be (8.74 +/- 0.26) x 10(3) M(-1) s(-1), whereas compound I reduction by o-dianisidine, pyrogallol and ascorbate was determined to be (2.71 +/- 0.03) x 10(6) M(-1) S(-1), (8.62 +/- 0.21) x 10(4) M(-1) S(-1), and (5.43 +/- 0.19) x 10(3) M(-1) S(-1), respectively. Cyanide binding studies on native and recombinant enzyme indicated that both have the same heme environment. An apparent second-order rate constant for cyanide binding of (4.8 +/- 0.1) x 10(5) M(-1) S(-1) was obtained.     

Paramagnetic carbon-13 NMR relaxation studies on the kinetics and mechanism of the HCO3-/CO2 exchange catalyzed by manganese(II) human carbonic anhydrase I

A detailed analysis of the stability and activity of Mn(II) human carbonic anhydrase I and the kinetics and mechanism of its catalysis of the HCO3-/CO2 exchange have been performed at pH 8.5. The analysis was based on the paramagnetic relaxation rates R1p and R2p of the 13C atom of HCO3- in the Mn2+/apoenzyme/HCO3-/CO2 system and the HCO3(-)----CO2 interconversion rate obtained by the magnetization-transfer technique. The R1p and R2p rates were measured as functions of the temperature, magnetic field strength, and substrate and apoenzyme concentrations and were interpreted on the basis of the Solomon-Bloembergen-Morgan theories and general equations for the ligand exchange [Led, J. J., & Grant, D. M. (1977) J. Am. Chem. Soc. 99, 5845-5858]. From the analysis of the data, a formation constant for the Mn(II) enzyme of log KMAM = 5.8 +/- 0.4 was obtained while the activity of the Mn(II) enzyme, measured as the HCO3-/CO2 interconversion rate at [HCO3-] = 0.100 M and pH 8.5, was found to be about 4% of that of the native Zn(II) enzyme. However, an effective dissociation constant KeffHCO3- less than or approximately 12 mM and a maximal exchange rate constant kcatexch approximately equal to 400 s-1, also derived by the analysis, result in an apparent second-order rate constant kcatexch/KeffHCO3- only a factor of 4 smaller than the corresponding rate constant for the native Zn(II) isoenzyme I. Most conspicuously, the resulting distance of only 2.71 +/- 0.03 A between the Mn2+ ion of the enzyme and the 13C atom of HCO3- in the enzyme-bicarbonate complex indicates that the bicarbonate is bound to the metal ion by two of its oxygen atoms in the central catalytic step, thereby supporting the modified Zn(II)-OH mechanism [Lindskog, S., Engberg, P., Forsman, C., Ibrahim, S. A., Jonsson, B.-H., Simonsson, I., & Tibell, L. (1984) Ann. N.Y. Acad. Sci. 429, 61-75 (and references cited therein)]. In contrast, this binding mode differs from the structure of the complexes suggested in the rapid-equilibrium kinetic model [Pocker, Y., & Deits, T. L. (1983) J. Am. Chem. Soc. 105, 980-986; Pocker, Y., & Deits, T. L. (1984) Ann. N.Y. Acad. Sci. 429, 76-83].     

Transferrins, the mechanism of iron release by ovotransferrin.

Iron release from ovotransferrin in acidic media (3 < pH < 6) occurs in at least six kinetic steps. The first is a very fast (相似文献   

Isotopic probes of the argininosuccinate lyase reaction

The mechanism of the argininosuccinate lyase reaction has been probed by the measurement of the effects of isotopic substitution at the reaction centers. A primary deuterium isotope effect of 1.0 on both V and V/K is obtained with (2S,3R)-argininosuccinate-3-d, while a primary 15N isotope effect on V/K of 0.9964 +/- 0.0003 is observed. The 15N isotope effect on the equilibrium constant is 1.018 +/- 0.001. The proton that is abstracted from C-3 of argininosuccinate is unable to exchange with the solvent from the enzyme-intermediate complex but is rapidly exchanged with solvent from the enzyme-fumarate-arginine complex. A deuterium solvent isotope effect of 2.0 is observed on the Vmax of the forward reaction. These and other data have been interpreted to suggest that argininosuccinate lyase catalyzes the cleavage of argininosuccinate via a carbanion intermediate. The proton abstraction step is not rate limiting, but the inverse 15N primary isotope effect and the solvent deuterium isotope effect suggest that protonation of the guanidino group and carbon-nitrogen bond cleavage of argininosuccinate are kinetically significant.     

Effects of denaturants at low concentrations on the reversible denaturation of staphylococcal nuclease

Three very unstable mutant forms of staphylococcal nuclease were used to quantitate the change in the apparent equilibrium constant for reversible denaturation (Kapp) as a function of denaturant concentration for a variety of different denaturing solutes. The value of this equilibrium constant in the absence of denaturant (Kapp,0) was determined by renaturation of the mutant proteins with a combination of glycerol and calcium ion, the latter of which binds at the active site in the native conformation. Because Kapp,0 fell in the easily measurable range between 0.1 and 1, the change in Kapp, and thus the change in free energy (delta Gapp), at very low concentrations of denaturants could be accurately measured. With guanidine hydrochloride (GuHCl), the rate of change of the apparent free energy of denaturation with respect to denaturant concentration (d(delta Gapp)/dCGuHCl or mGuHCl) was found to be remarkably constant down to zero denaturant concentration, even though this value was different for each of the three proteins. Unlike GuHCl, urea exhibited a slightly reduced value of d delta Gapp/dCurea at low concentrations. Results with a number of thiocyanate, perchlorate, and iodide salts confirmed that the Hofmeister series holds for concentrations below 0.1 M; that is, with regard to efficacy as a denaturant SCN- greater than ClO4- greater than I- and Li+,NH4+ greater than Na+,K+. However, all of the chaotropic salts analyzed exhibited markedly increased values of d(delta Gapp)/dCsalt at concentrations below 0.2 M. One possible explanation for these large deviations from a linear relationship between delta Gapp and salt concentration is that weak binding or adsorption of chaotropic anions is occurring at a saturable number of sites in hydrophobic regions of the denatured state.     

Effects of MgATP and MgADP on the cross-bridge kinetics of rabbit soleus slow-twitch muscle fibers.

The elementary steps surrounding the nucleotide binding step in the cross-bridge cycle were investigated with sinusoidal analysis in rabbit soleus slow-twitch muscle fibers. The single-fiber preparations were activated at pCa 4.40, ionic strength 180 mM, 20 degrees C, and the effects of MgATP (S) and MgADP (D) concentrations on three exponential processes B, C, and D were studied. Our results demonstrate that all apparent (measured) rate constants increased and saturated hyperbolically as the MgATP concentration was increased. These results are consistent with the following cross-bridge scheme: [cross-bridge scheme: see text] where A = actin, M = myosin, S = MgATP, and D = MgADP. AM+S is a collision complex, and AM*S is its isomerized form. From our studies, we obtained K0 = 18 +/- 4 mM-1 (MgADP association constant, N = 7, average +/- sem), K1a = 1.2 +/- 0.3 mM-1 (MgATP association constant, N = 8 hereafter), k1b = 90 +/- 20 s-1 (rate constant of ATP isomerization), k-1b = 100 +/- 9 s-1 (rate constant of reverse isomerization), K1b = 1.0 +/- 0.2 (equilibrium constant of isomerization), k2 = 21 +/- 3 s-1 (rate constant of cross-bridge detachment), k-2 = 14.1 +/- 1.0 s-1 (rate constant of reversal of detachment), and K2 = 1.6 +/- 0.3 (equilibrium constant of detachment). K0 is 8 times and K1a is 2.2 times those in rabbit psoas, indicating that nucleotides bind to cross-bridges more tightly in soleus slow-twitch muscle fibers than in psoas fast-twitch muscle fibers. These results indicate that cross-bridges of slow-twitch fibers are more resistant to ATP depletion than those of fast-twitch fibers. The rate constants of ATP isomerization and cross-bridge detachment steps are, in general, one-tenth to one-thirtieth of those in psoas.     

DNA tris-intercalation: first acridine trimer with DNA affinity in the range of DNA regulatory proteins. Kinetic studies

A trimer made up of three acridine chromophores linked by a positively charged poly(aminoalkyl) chain was synthesized as a potential tris-intercalating agent. The length of the linking chain was selected to allow intercalation of each chromophore according to the excluded site model. 1H NMR studies have shown that, at 5 mM sodium, pH 5, the acridine trimer occurred under a folded conformation stabilized by stacking interactions between the three aromatic rings. DNA tris-intercalation of the dye at a low dye/base pair ratio was shown by measurements of both the unwinding of PM2 DNA and the lengthening of sonicated rodlike DNA. The trimer exhibits a high DNA affinity for poly[d(A-T)] (Kapp = 8 X 10(8) M-1, 1 M sodium) as shown by competition experiments with ethidium dimer. Kinetic studies of both the association with poly[d(A-T)] and the exchange between poly[d(A-T)] and sonicated calf thymus DNA have been performed as a function of the ionic strength. In 0.3 M sodium the on-rate constant (k1 = 2.6 X 10(7) M-1 s-1) is similar to that reported for other monoacridines or bis(acridines), whereas the off-rate constant is much smaller (k-1 = 1.2 X 10(-4) s-1), leading to an equilibrium binding constant as large as Kapp = 2.2 X 10(11) M-1. A plot of log (k1/k-1) as a function of log [Na+] yielded a straight line whose slope shows that 5.7 ion pairs (out of 7 potential) are formed upon the interaction with DNA. From this linear relationship a Kapp value of 10(14) M-1 in 0.1 M sodium can be estimated. Such a value reaches and even goes beyond that of some DNA regulatory proteins. This acridine trimer appears to be the first synthetic ligand with such a high DNA affinity.     

Assessment by sedimentation equilibrium analysis of a heterologous macromolecular interaction in the presence of self-association: interaction of S5 with S8

The proteins S5 and S8 from the Escherichia coli 30S ribosomal subunit have been examined by sedimentation equilibrium methods for behavior in solution as isolated components and in mixtures. The means of resolving two simultaneous associations in this system is discussed, and the energy of association of S5 and S8 is reported. It was found that protein S5 from the MRE 600 strain tends to self-associate weakly at 4 degree C in a manner that can be described as an isodesmic self-association with an association constant and corresponding standard Gibbs free energy equal to (7.7 +/- 0.7) X 10(3) M-1 and -4.9 +/- 0.1 kcal/mol, respectively. Protein S8 was found to have a molecular weight of 15800 and was monomeric in a pure state. Mixtures of S5 and S8 clearly demonstrated the presence of an S5-S8 complex in addition to the self-association of S5. The equilibrium constant of association for the formation of a simple S5-S8 complex at 4 degree C and the corresponding standard Gibbs free energy were found to be (5.5 +/- 1.0) X 10(4) M-1 and -6.0 +/- 0.1 kcal/mol, respectively.     

Reassociation of dimeric cytoplasmic malate dehydrogenase is determined by slow and very slow folding reactions

Malate dehydrogenase occurs in virtually all eucaryotic cells in mitochondrial and cytoplasmic forms, both of which are composed of two identical subunits. The reactivation of the mitochondrial isoenzyme has been the subject of previous studies [Jaenicke, R., Rudolph, R., & Heider, I. (1979) Biochemistry 18, 1217-1223]. In the present study, the reconstitution of cytoplasmic malate dehydrogenase from porcine heart after denaturation by guanidine hydrochloride has been determined. The enzyme is denatured by greater than 1.2 M guanidine hydrochloride; upon reconstitution, approximately 60% of the initial native enzyme can be recovered. The kinetics of reconstitution after maximum unfolding by 6 M guanidine hydrochloride were analyzed by fluorescence, far-ultraviolet circular dichroism, chemical cross-linking with glutaraldehyde, and activity measurements. After fast folding into structured intermediates (less than 1 min), formation of native enzyme is governed by two parallel slow and very slow first-order folding reactions (k1 = 1.3 X 10(-3) S-1 and k2 = 7 X 10(-5) S-1 at 20 degrees C). The rate constant of the association step following the slow folding reaction (determined by k1) must be greater than 10(6) M-1 S-1. The energy of activation of the slow folding step is of the order of 9 +/- 1 kcal/mol; the apparent rate constant of the parallel very slow folding reaction is virtually temperature independent. The intermediates of reassociation must be enzymatically inactive, since reactivation strictly parallels the formation of native dimers. Upon acid dissociation (pH 2.3), approximately 35% of the native helicity is preserved, as determined by circular dichroism.(ABSTRACT TRUNCATED AT 250 WORDS)