Bose-Einstein condensation in biological systems

A theoretical model is presented for describing a previously untreated effect of viscosity on the apparent decomposition rate of enzyme-ligand complexes.Since the translational diffusion is hindered by the viscosity, its increased value results in an enlarged portion of ligands which can be rebound by the enzyme immediately after the dissociation of the complex.The model accounts for the experimentally observed decrease in maximal velocity of enzymic reactions at high viscosity. At the same time, it serves as a tool to obtain new information about the energetic processes of enzyme action.     

Effect of viscosity on enzyme-ligand dissociation. II. Role of the microenvironment

A theoretical treatment, describing a novel viscosity effect on decomposition of enzyme-ligand complexes, recently appeared (Somogyi et al., 1978). From this approach emerged a mechanistic picture of the manner in which increased viscosity lowers the value of the decomposition rate constant. A refined version of this model is presented herein. The analysis is extended to the molecular microenvironment ultimately responsible for mediating the "viscosity effect." Consideration is given to two major factors: (1) the role of viscosity in attenuating the excess chemical energy and (2) the statistical features of the microviscosity. In view of spatiotemporal inhomogeneity in the liquid structure, the concept of averaged microviscosity is introduced to parametrize the enzyme-ligand recombination probability. Quantitative predictions are consistent with models of liquid structure and with results from enzyme studies. The "viscosity effect" may contribute to substrate compartmentation in organized multi-enzyme systems in vivo.     

Influence of alpha-CH-->NH substitution in C8-CoA on the kinetics of association and dissociation of ligands with medium chain acyl-CoA dehydrogenase

We previously reported that the kinetic profiles for the association and dissociation of functionally diverse C(8)-CoA-ligands, viz., octanoyl-CoA (substrate), octenoyl-CoA (product), and octynoyl-CoA (inactivator) with medium chain acyl-CoA dehydrogenase (MCAD), were essentially identical, suggesting that the protein conformational changes played an essential role during ligand binding and/or catalysis [Peterson, K. L., Sergienko, E. E., Wu, Y., Kumar, N. R., Strauss, A. W., Oleson, A. E., Muhonen, W. W., Shabb, J. B., and Srivastava, D. K. (1995) Biochemisry 34, 14942-14953]. To ascertain the structural basis of the above similarity, we investigated the kinetics of association and dissociation of alpha-CH-->NH-substituted C(8)-CoA, namely, 2-azaoctanoyl-CoA, with the recombinant form of human liver MCAD. The rapid-scanning and single wavelength stopped-flow data for the binding of 2-azaoctanoyl-CoA to MCAD revealed that the overall interaction proceeds via two steps. The first (fast) step involves the formation of an enzyme-ligand collision complex (with a dissociation constant of K(c)), followed by a slow isomerization step (with forward and reverse rate constants of k(f) and k(r), respectively) with concomitant changes in the electronic structure of the enzyme-bound FAD. Since the latter step involves a concurrent change in the enzyme's tryptophan fluorescence, it is suggested that the isomerization step is coupled to the changes in the protein conformation. Although the overall binding affinity (K(d)) of the enzyme-2-azaoctanoyl-CoA complex is similar to that of the enzyme-octenoyl-CoA complex, their microscopic equilibria within the collision and isomerized complexes show an opposite relationship. These results coupled with the isothermal titration microcalorimetric studies lead to the suggestion that the electrostatic interaction within the enzyme site phase modulates the microscopic steps, as well as their corresponding ground and transition states, during the course of the enzyme-ligand interaction.     

cGMP-dependent protein kinase. Autophosphorylation changes the characteristics of binding site 1

cGMP-dependent protein kinase binds 4 mol cGMP/mol enzyme to two different sites. Binding to site 1 (apparent Kd 17 nM) shows positive cooperativity and is inhibited by Mg . ATP, whereas binding to site 2 (apparent Kd 100-150 nM) is non-cooperative and not affected by Mg . ATP. Autophosphorylation of the enzyme abolishes the cooperative binding to site 1 and the inhibitory effect of Mg . ATP. The association (K1) and dissociation (K-1) rate constant for site 2 and K1 for site 1 are not affected significantly by Mg . ATP or autophosphorylation. The dissociation rate from site 1 measured in the presence of 1 mM unlabelled cGMP is decreased threefold and over tenfold by Mg . ATP and autophosphorylation, respectively. In contrast, the dissociation rate from site 1 measured after a 500-fold dilution of the enzyme-ligand complex is 100-fold faster than that determined in the presence of 1 mM cGMP and is only slightly influenced by Mg . ATP or autophosphorylation. Only Kd values calculated with the latter K-1 values are similar to the Kd values obtained by equilibrium binding. These results suggest that autophosphorylation of cGMP-dependent protein kinase affects mainly the binding characteristics of site 1.     

Mechanism of azide binding to chloroperoxidase and horseradish peroxidase: use of an iodine laser temperature-jump apparatus

The kinetics of azide binding to chloroperoxidase have been studied at eight pH values ranging from 3.0 to 6.6 at 9.5 +/- 0.2 degrees C and ionic strength of 0.4 M in H2O. The same reaction was studied in D2O at pD 4.36. In addition, results were obtained on azide binding to horseradish peroxidase at pD 4.36 and pH 4.56. Typical relaxation times were in the range 10-40 microseconds. The value of kH/kD(on) for chloroperoxidase is 1.16, and kH/kD(off) is 1.7; corresponding values for horseradish peroxidase are 1.10 and 2.4. The H/D solvent isotope effects indicate proton transfer is partially rate controlling and is more important in the dissociation of azide from the enzyme-ligand complex. A mechanism is proposed in which hydrazoic acid binds to chloroperoxidase in a concerted process in which its proton is transferred to a distal basic group. Hydrogen bonding from the newly formed distal acid to the bound azide facilitates formation of hydrazoic acid as the leaving group in the dissociation process. The binding rate constant data, kon, can be fit to the equation kon = k3/(1 + KA/[H+]), where k3 = 7.6 X 10(7) M-1 S-1 and KA, the dissociation constant of hydrazoic acid, is 2.5 X 10(-5) M. The same mechanism probably is valid for the ligand binding to horseradish peroxidase.     

Interaction of phenols with old yellow enzyme. Physical evidence for charge-transfer complexes.

Evidence is presented that the changes in absorption spectrum obtained on complex formation between Old Yellow Enzyme and phenolic compounds are due to charge-transfer interactions. The positive correlation between the energy of the long wavelength transition and the Hammett para constant with a series of para-substituted phenols indicates that the phenol is the charge-transfer donor and the oxidized flavin of the enzyme is the charge-transfer acceptor. The same conclusion is drawn from studies in which the flavin of the native enzyme, flavin mononucleotide, was replaced by a variety of artificial flavins of different oxidation-reduction potential. The effect of pH on the dissociation constant for the enzyme-ligand binding also indicates that it is the phenolate anion, rather than the conjugate acid, which is responsible for the charge-transfer interaction. The significance of these results is discussed relative to long wavelength absorbing species detected with other flavoproteins.     

Detection and characterization of an additional site for binding of substrate and its analogs by inorganic pyrophosphatase

Phosphate, pyrophosphate, imidodiphosphate, EDTA and tripolyphosphate increase the rate constant for dissociation of the inorganic pyrophosphatase-substrate intermediate formed after cessation of the reaction by fluoride. The effect is enhanced in the given order 19-fold, the dependence of this effect on ligand concentration being hyperbolic. The values of the dissociation constants of the enzyme-ligand complexes lie within the concentration range of 0.16-1.0 mM. At high concentrations of Na2+ added simultaneously with the ligands this effect is decreased. The value of tau 1/2 for Pi binding to the enzyme-substrate compound is 0.15 min. The data obtained suggest that pyrophosphatase contains an anion ligand binding site, differing from that of the active one. This site does not affect the hydrolytic function of pyrophosphatase, as can be evidenced from the fact that Pi (9.5 mM) does not change the rate of enzymatic cleavage of PPi.     

Adenosine deaminase: viscosity studies and the mechanism of binding of substrate and of ground- and transition-state analogue inhibitors

We have studied the effects of viscosogenic agents, sucrose and ficoll, on (1) the hydrolysis of adenosine and of 6-methoxypurine riboside catalyzed by adenosine deaminase and (2) the rates of association and dissociation of ground-state and transition-state analogue inhibitors. For adenosine, Vmax/Km is found to be inversely proportional to the relative viscosity with sucrose, an agent affecting the microscopic viscosity, while no effect is found with ficoll, an agent affecting the macroscopic viscosity. Viscosogenic agents have no effect on the kinetic constants for 6-methoxypurine riboside. Thus, the bimolecular rate constant, Vmax/Km = 11.2 +/- 0.8 microM-1 s-1, for the reaction with adenosine is found to be at the encounter-controlled limit while that for the reaction with the poor substrate 6-methoxypurine riboside, 0.040 +/- 0.004 microM-1 s-1, is limited by some other process. Viscosity-dependent processes do not make a significant (less than 10%) contribution to Vmax. The dissociation constants for inhibitors are unaffected by viscosity. The ground-state analogue inhibitor purine riboside appears to bind at a rate comparable to that of adenosine. However, the slower rates of association (0.16-2.5 microM-1 s-1) and dissociation (5 X 10(-6) to 12 s-1) of transition-state analogue inhibitors are affected by the viscosity of the medium to approximately the same extent as the encounter-controlled rates of association and dissociation of adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic studies of Escherichia coli elongation factor Tu-guanosine 5'-triphosphate-aminoacyl-tRNA complexes

A new method for measuring the dissociation rate of the Escherichia coli elongation factor Tu-GTP--aminoacyl-tRNA complex has been developed and applied to the determination of the dissociation rates of ternary complexes formed between E. coli EF-Tu-GTP and a set of E. coli aminoacyl-tRNAs. The set of aminoacyl-tRNAs includes at least one tRNA coding for each of the 20 amino acids as well as purified isoacceptor tRNA species for arginine, glycine, leucine, lysine, and tyrosine. The results reveal that the dissociation rates vary for each ternary complex. Tu-GTP-Gln-tRNA dissociates the slowest and Tu-GTP-Val-tRNA the fastest of all noninitiator ternary complexes at 4 degrees C, pH 7.4. The equilibrium dissociation constant for Tu-GTP-Thr-tRNA has been determined to be 1.3 (0.4) X 10(-9) M under identical reaction conditions, and the absolute value of the equilibrium dissociation constant has been calculated for 28 ternary complexes from the relative equilibrium dissociation constant ratios previously measured [Louie, A., Ribeiro, N. S., Reid, B. R., & Jurnak, F. (1984) J. Biol. Chem. 259, 5010-5016]. The association rate of each ternary complex has been estimated from the ratio of the dissociation rate relative to the equilibrium dissociation constant. Tu-GTP-His-tRNA associates the fastest and Tu-GTP-Leu-tRNA1Leu the slowest. By inclusion of Tu-GTP-Met-tRNAfMet in the studies, evidence has been obtained that suggests that the initiator ternary complex does not function in the elongation cycle because the dissociation rate of the complex is very fast.     

On-chip non-equilibrium dissociation curves and dissociation rate constants as methods to assess specificity of oligonucleotide probes

Nucleic acid hybridization serves as backbone for many high-throughput systems for detection, expression analysis, comparative genomics and re-sequencing. Specificity of hybridization between probes and intended targets is always critical. Approaches to ensure and evaluate specificity include use of mismatch probes, obtaining dissociation curves rather than single temperature hybridizations, and comparative hybridizations. In this study, we quantify effects of mismatch type and position on intensity of hybridization signals and provide a new approach based on dissociation rate constants to evaluate specificity of hybridized signals in complex target mixtures. Using an extensive set of 18mer oligonucleotide probes on an in situ synthesized biochip platform, we demonstrate that mismatches in the center of the probe are more discriminating than mismatches toward the extremities of the probe and mismatches toward the attached end are less discriminating than those toward the loose end. The observed destabilizing effect of a mismatch type agreed in general with predictions using the nearest neighbor model. Use of a new parameter, specific dissociation temperature (T_d-w, temperature of maximum specific dissociation rate constant), obtained from probe–target duplex dissociation profiles considerably improved the evaluation of specificity. These results have broad implications for hybridization data obtained from complex mixtures of nucleic acids.     

Rotational diffusion studies of the lipoyl domain of 2-oxoacid dehydrogenase multienzyme complexes.

Rotational mobility of the lipoyl domain of a number of 2-oxoacid dehydrogenase complexes was investigated by transient dichroism after the domain had been specifically labeled with the triplet probe eosin-5-maleimide. Complexes investigated included pyruvate dehydrogenase complexes from Bacillus stearothermophilus, ox heart, and Escherichia coli (in which the E2 component had been genetically engineered to contain one lipoyl domain) and 2-oxoglutarate dehydrogenase complexes from ox heart and E. coli. Measurements were also performed with ox heart pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes specifically labeled on E1. Anisotropy decays were recorded in glycerol-buffer solutions of varying viscosity and at different temperatures. For E2-labeled complexes, the decays were found to be multiexponential, and the fastest correlation time was considerably shorter than expected for tumbling of the whole complex. This fast correlation time was absent from E1-labeled complexes and was assigned to independent motion of the lipoyl domain. Plots of the fast correlation time against eta/T showed a surprisingly weak dependence on viscosity and extrapolated to a time of 30-40 microseconds at zero viscosity. To explain this result, a model is proposed in which the lipoyl domain is in equilibrium between "free" and bound states. The time of 30-40 microseconds is shown to correspond to 1/koff, where koff is the rate constant for dissociation of the domain from binding sites on the complex. This dissociation phenomenon only contributes to the anisotropy decay when the viscosity of the solution is sufficiently high to slow the tumbling of the whole complex to times that are long in comparison to 1/koff.     

Analysis of trp repressor-operator interaction by filter binding.

A filter binding assay was developed that allows measurement of specific binding of trp repressor to operator DNA. The most important feature of this procedure is the concentration and type of salt present in the binding buffer. Using this assay the dissociation constant of the repressor-operator complex was determined to be 2.6 X 10(-9) M, and 1.34 repressor dimers were found to be bound to each operator-containing DNA molecule. These values agree with those obtained by more complex methods. The dissociation constant of the repressor for the corepressor L-tryptophan in the presence of operator DNA was shown to be 2.5 X 10(-5) M. A synthetic 48 bp operator fragment was used to determine the repressor-operator dissociation constant in the presence of tryptophan or tryptophan analogs which have higher or lower affinities for aporepressor. The rate of dissociation of repressor from operator DNA also was determined. Our findings indicate that dissociation is influenced by the concentration of tryptophan or tryptophan analogs and suggest that release of the corepressor may be the first step in dissociation of the repressor-operator complex.     

Basis for the anomalous effect of competitive inhibitors on the kinetics of hydrolysis of short-chain phosphatidylcholines by phospholipase A2.

The effect of four specific competitive inhibitors on the kinetics of hydrolysis of short-chain diacyl-sn-glycero-3-phosphocholines below their critical micelle concentrations was examined. The kinetics of hydrolysis of short-chain substrates dispersed as solitary monomers were generally consistent with the classical Michaelis-Menten formalism; i.e., hydrolysis began without any latency period, the steady-state rate was observed at higher substrate concentrations, the steady-state initial rate showed a linear dependence on the enzyme concentration, and the hyperbolic dependence of the initial rate on the substrate concentration could be described in terms of KM and Vmax parameters. The competitive nature of the inhibitors used in this study has been established by a variety of techniques, and the equilibrium dissociation constants for the inhibitors bound to the enzyme were measured by the protection method [Jain et al. (1991) Biochemistry 30, 7306-7317]. The kinetics of hydrolysis in the presence of competitive inhibitors could be described by a single dissociation constant. However, the value of the dissociation constant obtained under the kinetic conditions was comparable to that obtained by the protection method for the inhibitor-enzyme complex bound to a neutral diluent, rather than to the value of the dissociation constant obtained with solitary monomeric inhibitors and the enzyme in the aqueous phase. Spectroscopic methods showed that the effectively lower dissociation constant of an inhibitor bound to PLA2 at the interface is due to the stabilization of the enzyme-inhibitor complex by interaction with other amphiphiles present in the reaction mixture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic analysis of regeneration by dilution of a covalently modified protein.

An analysis of regeneration by dilution of a covalently modified protein is presented. It is shown that, when protein regeneration is realized through the intermediacy of a protein-modifying agent adsorptive complex, the reaction is described by a summation of two exponential functions of reaction time plus a constant-term equation. The conditions whereby this equation reduces to a single-exponential equation are delineated. It is shown that, when protein regeneration is described by a single-exponential function of reaction time, the first-order protein-regeneration rate constant is a function of modifying-agent concentration and also of the microscopic reaction rate constants. Accordingly, the protein-modifying agent dissociation constant (Ki), as well as the protein-covalent-modification and -regeneration, rate constants (k+2 and K-2), may be determined by an analysis of dilution-induced protein-regeneration (or enzyme-reactivation) data obtained at different dilutions of the covalently modified protein-modifying agent preparation.     

A Theoretical and Experimental Study of Competition Between Solution and Surface Receptors for Ligand in a Biacore Flow Cell

Rate constants that characterize the kinetics of binding and dissociation between biomolecules carry fundamental information about the biological processes these molecules are involved in. An instrument that is widely used to determine these rate constants is the Biacore. In a Biacore experiment, one of the reactants, which we will call the receptor, is immobilized on a sensor chip. During the binding phase of the experiment the other reactant flows past the chip. After binding, buffer alone is introduced into the flow cell and dissociation is monitored. Often surface-based binding assays are influenced by the transport of the reactant in solution, complicating the determination of the chemical rate constants from the observed binding kinetics. We propose a new way to determine the dissociation rate constant by adding soluble receptor during dissociation. The method is tested first on simulated data and then on Biacore experiments where the lac repressor protein binds and dissociates from a stretch of double stranded DNA containing the lac repressor binding site. With this method we find a dissociation rate constant k_d=0.075 ± 0.005s^-1, a value that is faster than previously obtained from Biacore experiments. In developing our method to analyze these experiments we obtain an expression for the transport limited rate constant for a Biacore experiment when soluble receptor is present during dissociation.     

Anion effects on the liver alcohol dehydrogenase reaction

The effects of various anions on the rate constant for dissociation of NADH from a binary complex with horse liver alcohol dehydrogenase were evaluated. Phosphate, sulfate, and fluoride had no effect, while nitrate and the other halide ions caused a three- to fourfold increase in the rate constant for NADH dissociation. These results indicate that a ternary enzyme-NADH-anion complex is formed, and from the anion concentration dependence the relative affinities are iodide greater than nitrate and bromide greater than chloride. At high salt concentrations, above 0.2 M, the rate constants for NADH dissociation decreased, which was attributed to a decrease in the activity coefficient of the reactants or "salting in." The rate constant for NADH dissociation from ternary complex with imidazole, which crystallizes in an orthorhombic form rather than triclinic, was also substantially enhanced by anions. This provides an indication that the enhancement is independent of the conformational state of the enzyme complex. Thus, the most likely explanation for the observed enhancement of NADH dissociation is anion interference with binding of the coenzyme pyrophosphate group, which does not occur with larger anions such as phosphate or sulfate. Since NADH dissociation partially limits the turnover of the enzyme, the effect of nitrate on steady-state turnover was determined. A twofold increase was observed at optimal levels of nitrate, at both substrate inhibitory and noninhibitory concentrations of ethanol.     

Limiting rates of ligand association to alcohol dehydrogenase.

Detailed stopped-flow kinetic studies of the association of 2,2-bipyridine, 1,10-phenanthroline, and 5-chloro-1,10-phenanthroline to the zinc ion at the active site of alcohol dehydrogenase have demonstrated that a process with a limiting rate constant of about 200 s^?1 restricts the binding of the bidentate chelating agents to the free enzyme. The formation of the enzyme-ligand complexes has been followed by means of the characteristic absorption spectra of the resulting complexes or by the displacement of the fluorescent dye, auramine O. Monodentate ligands, upon binding to the free enzyme or enzyme-NAD⁺ and enzyme-NADH complexes, do not exhibit a comparable limiting rate. In analogy with simple inorganic systems, these observations have been interpreted in terms of the rate limiting dissociation of an inner sphere water molecule following the rapid formation by the bidentate ligand of an outer sphere complex. The displacement of a water molecule from the zinc ion by 1,10-phenanthroline has been observed in crystallographic studies which have also established that the zinc ion in the enzyme-1,10-phenanthroline complex is pentacoordinate. Monodentate ligands, which are substrate analogs, do not exhibit limiting rates because displacement of water is not required for their addition to a coordinate position which is apparently vacant in the free enzyme. If a water molecule remains bound to the zinc ion in the kinetically competent ternary complex, it could play an essential role in the proton transfer reaction accompanying catalysis.     

Transient kinetic studies of substrate inhibition in the horse liver alcohol dehydrogenase reaction

The rate-limiting step of ethanol oxidation by alcohol dehydrogenase (E) at substrate inhibitory conditions (greater than 500 mM ethanol) is shown to be the dissociation rate of NADH from the abortive E-ethanol-NADH complex. The dissociation rate constant of NADH decreased hyperbolically from 5.2 to 1.4 s-1 in the presence of ethanol causing a decrease in the Kd of NADH binding from 0.3 microM for the binary complex to 0.1 microM for the abortive complex. Correspondingly, ethanol binding to E-NADH (Kd = 37 mM) was tighter than to enzyme (Kd = 109 mM). The binding rate of NAD+ (7 X 10(5) M-1s-1) to enzyme was not affected by the presence of ethanol, further substantiating that substrate inhibition is totally due to a decrease in the dissociation rate constant of NADH from the abortive complex. Substrate inhibition was also observed with the coenzyme analog, APAD+, but a single transient was not found to be rate limiting. Nevertheless, the presence of substrate inhibition with APAD+ is ascribed to a decrease in the dissociation rate of APADH from 120 to 22 s-1 for the abortive complex. Studies to discern the additional limiting transient(s) in turnover with APAD+ and NAD+ were unsuccessful but showed that any isomerization of the enzyme-reduced coenzyme-aldehyde complex is not rate limiting. Chloride increases the rate of ethanol oxidation by hyperbolically increasing the dissociation rate constant of NADH from enzyme and the abortive complex to 12 and 2.8 s-1, respectively. The chloride effect is attributed to the binding of chloride to these complexes, destabilizing the binding of NADH while not affecting the binding of ethanol.     

Effects of the ligands of beef tryptophanyl-tRNA synthetase on the elementary steps of the tRNA(Trp) aminoacylation

Tryptophanyl-tRNA synthetase catalyzed formation of Trp-tRNA(Trp) has been studied by mixing tRNA(Trp) with a preformed bis(tryptophanyl adenylate)-enzyme complex in the 0-60-ms time range, on a quenched-flow apparatus. Analyzing the data gives an association rate constant ka = (1.22 +/- 0.47) X 10(8) M-1 S-1, a dissociation rate constant kd = 143 +/- 73 S-1, and a dissociation constant Kd = 1.34 +/- 0.80 microM for tRNA(Trp). The maximum rate constant of tryptophan transfer to tRNA(Trp) is kt = 33 +/- 3 S-1. When starting the aminoacylation reaction with a mono(tryptophanyl adenylate)-enzyme complex, one obtains different kinetic profiles than when using a bis(tryptophanyl adenylate)-enzyme complex. Over a 0-400-ms time range, the monoadenylate-enzyme complex yields an apparent first-order reaction, while the bis-adenylate-enzyme complex yields a biphasic aminoacylation of tRNA(Trp). Analysis of Trp-tRNA(Trp) formation from both complexes according to simple reaction schemes shows that the dissociation of tRNA(Trp) from an enzyme subunit carrying no adenylate is 6.9-fold slower than from an enzyme subunit carrying an adenylate. The apparent rate constant of dissociation of nascent tryptophanyl-tRNA(Trp) is 4.9 S-1 in the absence of free tryptophan, which is much slower than its rate of formation (33 S-1).(ABSTRACT TRUNCATED AT 250 WORDS)