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M R Eftink

Publications and source records attributed to M R Eftink.

66 records · Page 4Linked to original sources
PubMed1983

Energetics of ribonuclease A catalysis. 3. Temperature dependence of the hydrolysis of cytidine cyclic 2',3'-phosphate.

Studies of the temperature dependence of the steady-state kinetics of the ribonuclease A catalyzed hydrolysis of cytidine cyclic 2',3'-phosphate at pH 5.0 are reported. Contributions to the temperature dependence of the apparent Michaelis-Menten parameters from temperature-sensitive protonic equilibria (primarily the coupled protonation/deprotonation of the active-site histidine residues) were included in our analysis. The data were interpreted by employing a transition-state approach. By comparing the temperature dependence of the rate constant for the nonenzymatic hydrolysis of the substrate with the temperature dependence of the enzyme-catalyzed reaction, we obtained values for the enthalpy change, entropy change, and heat capacity change for the interaction of the reaction transition state with the enzyme. These thermodynamic quantities were then interpreted by comparison with corresponding values for the binding of cytidine 2'- and 3'-phosphate to the enzyme. A model is presented for the enzyme-transition-state interaction involving the favorable transfer of a proton from the transition state to a histidine residue at the active site and the formation of hydrogen bonds and van der Waals contacts between the pyrimidine ring of the transition state and the enzyme's binding pocket. These elementary interactions are consistent with the determined values of the enthalpy change and entropy change, as well as earlier reported ionic strength and solvent isotope dependence studies. The Gibbs energy contributions from these elementary interactions have also been estimated, giving a sum approximately equal to the experimentally determined value for the stabilization energy of the enzyme-transition-state complex. The model thus provides an explanation for the magnitude of the approximately 10(10)-fold rate enhancement achieved by this enzyme.

Catalysis↗
PubMed1983

Enthalpy-entropy compensation and heat capacity changes for protein-ligand interactions: general thermodynamic models and data for the binding of nucleotides to ribonuclease A.

General thermodynamic models are presented that can account for the existence of heat capacity changes and compensation between the enthalpy and entropy changes in protein-ligand interactions. The models involve the coupling between some type of transition in the state of the protein (or ligand) and the binding process. The coupled transition may be a proton dissociation, the binding of a second ligand, a change in the degree of aggregation, or a conformational change in either the protein or ligand. Both mandatory coupling and nonmandatory coupling between the binding process and the transition are considered. The model is also extended to include a multistate transition of the protein. Computer simulations show that apparently linear compensation plots (plots of delta H degrees vs. delta S degrees) with a slope approximately equal to the experimental temperature are to be expected for the binding of a ligand to a protein when such coupled reactions exist. Also heat capacity changes, which may be either positive or negative, are to be expected to accompany the reaction. Experimental thermodynamic data for the binding of cytidine 3'-phosphate to ribonuclease A are presented. These data demonstrate apparent enthalpy-entropy compensation when pH and ionic strength are varied. A negative heat capacity change, ranging from -145 (at mu = 1.0 M) to -225 cal/(mol X deg) (at mu = 0.05 M), is also observed for this protein-ligand interaction. The apparent compensation and heat capacity change data are interpreted according to the models presented.

Computers↗
PubMed1982

Acrylamide and oxygen fluorescence quenching studies with liver alcohol dehydrogenase using steady-state and phase fluorometry.

The fluorescence lifetime of liver alcohol dehydrogenase (LADH) has been determined by phase fluorometry at various emission wavelengths and as a function of the concentration of the quencher acrylamide. Acrylamide selectively quenches the fluorescence of the surface tryptophanyl residue Trp-15, thus allowing the fluorescence lifetime of this residue and the buried residue Trp-314 to be evaluated. Values of tau15 = 6.9 ns and tau314 = 3.6 ns are obtained, in qualitative agreement with lifetimes of these residues determined from fluorescence decay studies [Ross, J.B.A., Schmidt, C.J., & Brand, L. (1981) Biochemistry 20, 4369-4377]. The quenching of the fluorescence of LADH by oxygen has also been studied. Quenching by oxygen results in a blue shift in the fluorescence of the protein and a downward-curving Stern-Volmer plot. These data, along with oxygen quenching studies in the presence of 1 M acrylamide, are consistent with a model in which oxygen quenches the fluorescence of Trp-314 and -15 with quenching constants of 3.5 and 25 M-1, respectively. Thus, as in studies with other quenchers, Trp-314 is found to be less accessible to the quencher oxygen than is Trp-15. A lifetime Stern-Volmer plot has also been obtained for the oxygen quenching of LADH. Such a plot deviates somewhat from the intensity Stern-Volmer plot as predicted by simulations of the quenching of two-component systems.

Acrylamides↗
PubMed1982

Fluorescence quenching of liver alcohol dehydrogenase by acrylamide.

The quenching of the fluorescence of liver alcohol dehydrogenase by acrylamide has been studied as a function of excitation and emission wavelength. Downward curving Stern-Volmer plots are found, providing further support for the notion that Trp-15 of the protein is surface exposed and that Trp-314 is extensively buried within the protein. The acrylamide quenching of the binary complex formed between the protein and NAD+ was also studied. The quenching pattern in this case is found to be complicated due to the interaction of acrylamide with the binary complex. Independent evidence for the fact that acrylamide binds to the binary complex is obtained from enzyme inhibition studies and from NAD+ binding studies showing acrylamide to bind with positive cooperativity with respect to the coenzyme. When the interaction of acrylamide with the binary complex is taken into consideration, however, the quenching data can be interpreted as indicating that the binding of NAD+ to the protein does not induce a conformational change that leads to the exposure of Trp-314.

Acrylamides↗
PubMed1977

Exposure of tryptophanyl residues and protein dynamics.

The acrylamide quenching reaction is shown to be very discriminating in sensing the exposure of fluorescing tryptophanyl residues in globular proteins. The quenching rate constants for some proteins, such as aldolase and human serum albumin, are reported to be independent of the solvent viscosity, indicating that the reaction is limited by penetration of the quencher through the protein matrix. Temperature-dependent studies are performed to determine the activation energy and entropy for the penetration of acrylamide into these proteins. The tryptophanyl residues in aldolase are shown to be shielded by a large activation energy barrier, while the single residue in human serum albumin is shielded by a large activation entropy barrier. These parameters characterize the nature of the protein matrix enveloping the fluorophors.

Acrylamides↗
PubMed1977

A hydrophobic quencher of protein fluorescence: 2,2,2-trichloroethanol.

Previously the neutral fluorescence quenching probe, acrylamide, was employed to determine the degree of exposure of tryptophan residues in proteins. A less polar neutral quencher 2,2,2-trichloroethanol (trichloroethanol) was used in the present work to investigate whether it would preferentially interact with apolar regions of proteins. For most proteins studied, the degree of quenching by trichloroethanol is found to be about the same as with acrylamide. However, for human and bovine serum albumin hydrophobic interactions between trichloroethanol and these proteins occur, leading to an exalted quenching. The fluorescence quencher thus senses the presence of a hydrophobic domain in the vicinity of the tryptophan residues in these proteins. Trichloroethanol is shown to induce conformational changes in certain proteins and to be a potentially useful quencher for proteins having predominantly tyrosine emission.

Acrylamides↗
PubMed1976

Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies.

Acrylamide is an efficient quencher of tryptophanyl fluorescence which we report to be very discriminating in sensing the degree of exposure of this residue in proteins. The quenching reaction involves physical contact between the quencher and an excited indole ring, and can be kinetically described in terms of a collisional and a static component. The rate constant for the collisional component is a kinetic measure of the exposure of a residue in a protein, and values ranging from 4 X 10(9) M-1 S-1 for the fully exposed tryptophan in the polypeptide, adrenocorticotropin, to less than 5 X 10(8) M-1 S-1 for the buried residue in azurin have been found. Static quenching is readily detected in proteins that are denatured, or contain only a single fluorophor. Quenching patterns for most multi-tryptophan containing proteins are difficult to analyze precisely, but qualitative information can, nevertheless, be extracted. Applications of this probing technique for monitoring protein conformational changes, such as the acid-induced expansion of human serum albumin, and inhibitor binding to enzymes, are presented. The value of this method lies in its ability to sense not only the steady-state exposure of a residue in a protein, but also its dynamic exposure.

Acrylamides↗
PubMed1975

Dynamics of a protein matrix revealed by fluorescence quenching.

The fluorescence of the supposedly buried tryptophan in ribonuclease T1 has been found to be collisionally quenched by acrylamide with a rate constant of 3 X 10(8) M--1 sec--1. Only a slight decrease in the quenching rate is observed upon a 5-fold increase in the viscosity of the solution. For this to be the case, the diffusion of the quencher must be limited by the protein matrix. To explain the process of diffusion through this complex material, the formation of "holes" in the lattice of a protein due to nanosecond fluctuations must be invoked. Thus, the dynamic character of a protein molecule is revealed. The quenching rate constant has an activation energy of 9 kcal/mol which can be used to characterize the nature of the cohesive forces in the microenvironment about the indole ring. The mechanical properties of a portion of a protein matrix can, therefore, be described as one would for a fluid.

Acrylamides↗