Rate constants determined by nuclear magnetic resonance

Fast kinetic methods are used to measure reactions that take place in less time than required to mix the reagents manually and to measure the reaction by usual methods, like UV-visible spectrophotometry and fluorescence. The best known of them are rapid-mixing and relaxation methods, which are used for reactions with half-times in the millisecond and microsecond ranges, respectively. The picosecond range is usually measured with electrical field and ultrasonic waves (A. Cornish-Bowden, 1976, Principles of Enzyme Kinetics, pp. 164-167, Butterworths, London). Normally these very fast rates occur when a ligand binds to or dissociates from a protein. When the binding is mediated only by the diffusion, the lower limit of the association rate constant (kon) should not exceed the value of a diffusion-controlled reaction (around 1010 M-1 s-1). Therefore, the values most frequently found for these rate constants, for example, in the association of a substrate with an enzyme, are in the range 106 to 109 M21 s21 (M. Eigen and G. G. Hammes, 1963, Adv. Enzymol. 25, 1–38). The values forthe dissociation rate constants (koff) for these reactions, which depend on the equilibrium constant for the enzyme–substrate complex interaction, are in the range 101 to 105 s21, most often between 103 and 104 s21 (A. Fersht, 1999, Structure and Mecha-nism in Protein Science, pp. 164–165, Freeman, New York). If the equilibrium constant is known, and the value of koff is determined by nuclear magnetic resonance (NMR), as described in this chapter, the value of kon can be calculated; this should not exceed mothe value of diffusion rate in the media in which the reaction is performed

Applications of nuclear magnetic resonance to determine the structure and interactions of ligands, peptides and enzymes

Nuclear magnetic resonance (NMR) spectroscopy is emerging as a powerful tool for the study of enzyme structure and function. This article discusses the general principles of NMR and the potential information this technique can provide in the study of enzymes along with its limitations

Inhibition of Tubulin Self-Assembly and Tubulin-Colchicine GTPase Activity by Guanosine 5’-γ-Fluorotriphosphate)

The inhibitory effects of guanosine 5'-(ᵧ-fluorotriphosphate) [GTP(ᵧF)] on both the polymerization and the colchicine-dependent GTPase activity of calf brain tubulin have been studied. The results demonstrate that this analogue of GTP, with a fluorine atom on the ᵧ-phosphate, is a reversible competitive dead-end inhibitor of the colchicine-induced GTPase activity with a K1 value of (1.8 ± 0.6) × 10-4 M. GTP(ᵧF) did not promote assembly of tubulin from which the E-site guanine nucleotide had been removed. It binds to the exchangeable nucleotide site competitively with respect to GTP, diminishing both the rate and extent of tubulin polymerization. Treatment in terms of the Oosawa-Kasai model of the inhibitory effect of GTP(ᵧF) on the assembly led to a value of Kdis = 1.1 × 10-6 M for the complex GTP(ᵧF)-tubulin. This analogue does not bind to the postulated third site. The growing of tubulin polymers at 37 °C was arrested by GTP(ᵧF), and only limited depolymerization was induced by the

Initiator-like properties of a methionyl-tRNA from wheat embryos

The two major methionyl-tRNA species from wheat embryos and E. coli have been studied as regards their capacity to form a ternary complex with GTP and the ribosomal binding enzyme from both sources. Methionyl-tRNA1 from wheat resembles met-tRNAF from E. coli in its inability to interact with the homologous enzyme. It also fails to complex with the bacterial enzyme. Methionyl-tRNA2 from wheat is similar to met-tRNAM from E. coli in that forms the ternary complex with the enzyme from both organisms. Wheat met-tRNA1 has a markedly higher affinity for binding "non-enzymatically" to wheat ribosomes in the presence of ApUpG than does met-tRNA2. © 1970

Calcium and gadolinium ions stimulate the GTPase activity of purified chicken brain tubulin through a conformational change

Ca2+ and Gd3+ stimulated the GTPase activity of chicken brain tubulin 13- and 26-fold, respectively. Mg2+, Tb3+, and Na+ had no effect. This GTPase activity showed a saturation behavior with Ca2+ and Gd3+ with a maximal activity of 0.26 ± 0.026 and 1.15 ± 0.78 nmol min-1 per mg of tubulin and semisaturation constants, expressed as the concentration of the cation needed for 50% of saturation, of 0.32 ± 0.18 and 0.011 ± 0.007 mM, respectively. In the presence of Ca2+, the GTPase activity was proportional to tubulin concentration in the range 0.9-31.8 μM. The semisaturation constants for the inhibition of tubulin polymerization and for the depolymerization of microtubules by Ca2+ were 0.71 ± 0.1 and 0.049 ± 0.043 mM, respectively. The similarity of the Ca2+ semisaturation constants for inhibition of tubulin assembly and stimulation of the GTPase activity suggests that these processes are correlated. These results support the hypothesis that the GTPase activity is related to but not direc

Interaction between the C-Terminal Peptides of Tubulin and Tubulin S Detected with the Fluorescent Probe 4',6-Diamidino-2-Phenylindole

The digestion of tubulin with subtilisin and the reassociation of the digestion products was followed by means of the fluorescent probe 4',6-diamidino-2-phenylindole (DAPI). The fluorescence spectra of DAPI bound to chicken brain tubulin and to the main products of tubulin digested with subtilisin-agarose (tubulin S and C-terminal peptides) were analyzed. The corrected emission spectrum of DAPI in the presence of tubulin showed an enhancement of fluorescence intensity with a maximum at 452 nm. The digestion reaction was followed by the diminution of the area of DAPI-tubulin emission spectra, which showed biphasic pseudo-first-order kinetics. The values for the rate constants were 1.2 x 10-2 min-1 and 3.5 x 10-2 min-1 for the α and β subunits, respectively, and were similar to those determined from the undigested subunits using polyacrylamide gel electrophoresis. Tubulin S and the C-terminal peptides were purified by means of a Bio-Gel P-60 column. The C-terminal peptides obtained fro

Corrigendum to “Overcoming electrostatic repulsions during amyloid assembly: Effect of pH and interaction with divalent metals using model peptides” [Arch. Biochem. Biophys. 621 (2017) 46–53]

research grants CONICYT PAI/CONCURSO NACIONAL INSERCION EN LA ACADEMIA, CONVOCATORIA 2016 PAI79160143 CONICYT/FONDECYT/3150054 CONICYT/FONDECYT/1116055