History of advances in enzyme kinetic methods: From minutes to milliseconds.

The auxin-induced growth reaction of the Avena coleoptile has been treated by methods of classical enzyme kinetics. The kinetic treatment makes it possible to characterize the growth promoting activity of an auxin by two parameters, K[subscript s] and V[subscript max]. These express respectively the affinity of the auxin for the auxin-receptive site within the plant and the ability of the complex thus established to promote growth. Treatment of Avena section growth by the methods of enzyme kinetics has made it possible to determine rigorously whether or not inhibitors of auxin-induced growth are true antiauxins and act by competing with auxin for the auxin-receptive site within the plant. Certain auxin-inactive compounds have been shown to possess antiauxin activity. Among such substances are 2,4-dichloroanisole, 4,-chloro- and 2,4-dichlophenoxyisobutyric acids, and 2,6-dichloro-and 2,4,6-trichlorophenoxyacetic acids. Each of these substances can be considered as derived from an active auxin (2,4-dichlorophenoxyacetic acid) by elimination of one of the structural features essential to auxin activity and thereby capable of combining at one point of the auxin-receptive site but incapable of consumating the two-point attachment requisite for auxin activity. It is shown that chemically different auxins compete with one another for the same receptive sites within the plant. Auxins of low v[subscript max] are capable of inhibiting or augmenting the activity of auxins of greater V[subscript max]. Whether inhibition or promotion result depend on the concentrations of the two substances and the differential in V[subscript max] exhibits apparent antiauxin activity. This activity is shown to be different from competitive inhibition of true antiauxins. The relationship between Avena section growth rate and auxin concentration has been demonstrated to be predictable on the basis of a requirement for two-point attachment of the auxin molecule to some receptive entity within the plant. The growth inhibition resulting from high auxin concentrations is not alleviated by antiauxins but rather the auxin inhibition is augmented by the presence of sufficiently high antiauxin concentrations. A necessary corollary to the single point attachment antiauxin concert and the bimolecular complex formation concept for inhibitory auxin concentrations is therefore confirmed. A preliminary investigation concerning herbicidal activity of mixtures of an antiauxin and an auxin on bean plants is presented. The data obtained do not unequivocally establish that inactive bimolecular auxin receptor complex formation at high auxin concentrations is a factor contributing to herbicidal action of 2,4-D, but the possibility that this is in fact so is considered. A cultural technique for obtaining isolated flax root clones is described and data for some experiments with an isolated flax root clone are presented. The inhibitory action of certain antimetabolites on the growth of young tomato plants is described.

To analyze the performance characteristics, stability, and clinical value of lipoprotein-associated phospholipase A2 (Lp-PLA2) using an enzymatic kinetic method. The performance characteristics included reference intervals, precision, and accuracy. We assessed Lp-PLA2 stability by comparing Lp-PLA2 changes under different conditions. Lp-PLA2 was determined in the following groups: control individuals, patients with coronary heart disease (CHD), patients of different lipid subgroups within CHD, and patients with high total cholesterol (TC). Also, correlations between Lp-PLA2 and traditional cardiovascular risk factors were assessed. The mean (SD) reference interval of serum Lp-PLA2 activity was 451 (113) U per L with sex differences. Inter- and intra-assay precision revealed coefficients of variance (CVs) of 1.81% to 2.63% and 1.43% to 1.77%. The average bias was 0.33%. Lp-PLA2 activity was stable. In the CHD group, high-lipid subgroups, and high-TC group, Lp-PLA2 was elevated, and correlation was observed between Lp-PLA2 and traditional risk factors. Lp-PLA2 activity has important clinical value in CHD.

Analysis of 5&-reductase(5&R) is commonly done by measurement of distinct testosterone(T) metabolites, following tissue incubation. Interlaboratory comparison is difficult, as different individual methods are used. In order to overcome this disadvantage, 5&R analysis was performed by evaluation of enzyme kinetics, according to Michaelis-Menten (Km- and Vmax-values), and by comparing the results with a conventional method. 17 tissue specimens (foreskin) of healthy boys (1 to 8 yrs) were examined with two methods (A and B). A: Conventional assay, using 8 pmol 3H-T as substrate. B: Evaluation of enzyme kinetics with increasing concentrations of 3H-T (8 to 208 pmol). Separation of metabolites by thin layer chromatography (TLC). Zone detection by computing radio TLC scanner. Control of purity and specific radioactivity by high resolution radio gas chromatography. Results showed age dependent values of 5&R (x̄+SEM): Vmax=18.49+2.57 pmol/mg.h; Km= 102.82+11.41 nM, with a maximum at age 5 yrs: Vmax=36.33+4.32 pmol/mg.h; Km=171.77+23.53 nM. Conclusion: It is recommended to evaluate 5&R-analysis by the enzyme kinetic method. The results of Vmax correspond to those obtained with the single point assay (method A, r=0.98). The advantage of method B is a reproduceable method giving biochemically absolute data for quality and quantitiy of the enzyme.

Symbols and Abbreviations Introduction Multiple Equilibria - Diffusion - Interaction of ligands with macromolecules o Macromolecules with identical, non-identical, independent and interacting binding sites Enzyme Kinetics - reaction order - kinetics, o analysis of kinetic data, enzyme inhibition, multi-substrate reactions, o analysis of complex enzyme mechanisms, allosteric enzymes, - PH, temperature dependence of enzymes - Isotope exchange - Statistical methods in enzyme kinetics Methods - methods for the investigation of multiple equilibria, electrochemical methods, spetroscopy, calorimetry, measurements of fast reactions Index

Exploration of drug action on a morphine receptor by methods of enzyme kinetics

The interaction of D-xylose isomerase purified from two sources with Mn2+ and D-xylose or the competitive inhibitor xylitol has been examined by nuclear magnetic resonance. A greater paramagnetic effect of enzyme-bound Mn2+ on the alpha anomer of D-xylose than on the beta anomer was observed, providing independent evidence for the specificity of D-xylose isomerase for the alpha anomeric form of D-xylose. The exchange rate of alpha-D-xylose into the ternary complex, determined from the normalized paramagnetic contribution to the transverse relaxation rate (1/fT2p) of the carbon 1 proton of alpha-D-xylose, exceeds Vmax for the enzymatic reaction by 3 orders of magnitude. The amount of xylitol necessary to displace alpha-D-xylose from the substrate-enzyme-Mn2+ complex is consistent with the Km value for alpha-D-xylose and the inhibitor constant Ki for xylitol previously determined by the methods of enzyme kinetics. These results suggest that the NMR experiments observe complexes of D-xylose isomerase which are kinetically and thermodynamically competent to participate in catalysis. From the frequency dependence of the paramagnetic contribution to the longitudinal relaxation rate (1/T1p) of the carbon 1 proton of alpha-D-xylose, the correlation time (tauc) which modulates the dipolar interaction between enzyme-bound Mn2+ and alpha-D-xylose has been determined (5.1 x 1o(-10) s). From these observations a range of calculated distances between enzyme-bound Mn2+ and the carbon 1 proton of alpha-D-xylose (9.1 +/- 0.7 A) has been found. The enzyme-bound Mn2+ has comparable effects on the carbon 1, carbon 2, and carbon 5 protons of alpha-D-xylose, suggesting that these protons of the enzyme-bound substrate are equidistant from the bound Mn2+. A similar distance (9.4 +/- 0.7 A) between the enzyme-bound Mn2+ and the terminal methylene protons of xylitol, an analog of the open chain intermediate in the reaction, has been determined. The results of the present substrate relaxation and previous water relaxation studies suggest that two small ligands such as water molecules or a large portion of the protein intervene between the bound metal ion and the bound substrate in the active ternary complex.

Synthesis, molecular docking, and biological activity of 2-vinyl chromones: Toward selective butyrylcholinesterase inhibitors for potential Alzheimer's disease therapeutics

At some point in characterizing an enzymatic reaction mechanism, kinetic information is required. This may range from the evaluation of the substrate specificity through comparison of Michaelis-Menten kinetic parameters Km and Vm for various substrates to the elucidation of the complete kinetic mechanism and evaluation of rate constants for all the steps. In this chapter, we outline the theory and methods of enzyme kinetics and show for a few simple cases the mechanistic information that can be derived. The steady-state kinetic analysis of enzymatic reactions nearly always entails the measurement of initial rates as a function of varying concentrations of a substrate at a fixed enzyme concentration. An initial rate best represents enzyme activity because it is the rate at time zero, before any of the many factors that can decrease enzyme activity come into play. These factors include inhibition by products, changes in pH, denaturation of the enzyme and so forth. In chemical kinetics, a large fraction of the time course for the reaction is usually measured to obtain a large number of data points to determine the kinetic order of the reaction. No problems with denaturation and product inhibition complicate such measurements. In contrast, the progress curve for an enzymatic reaction is generally sensitive to the accumulation of products, which are inhibitory and have to be taken into account. Moreover, the activity of an enzyme sometimes changes because of instability or environmental factors. However, accurate and reproducible initial rates can generally be obtained. It is possible to follow the full course of an enzymatic reaction by measuring the progress curve, as in conventional chemical kinetics. By fitting the curves to the integrated rate equations the steady-state kinetic parameters for an enzyme can be obtained from a single progress curve (Duggleby, 1995). This method has a number of advantages in principle. In practice, however, the many complications mentioned earlier, especially enzyme stability under reaction condition, have led kineticists to favor the measurement of initial rates at varying substrate concentrations in steady-state kinetic analysis.

Biochemical and structural insights into PLP fold type IV transaminase from Thermobaculum terrenum

Interaction of AR and iNOS in lens epithelial cell: A new pathogenesis and potential therapeutic targets of diabetic cataract

Principles of selectivity of sodium and potassium binding sites of the Na +/K +-ATPase. A corollary hypothesis

Synthesis, molecular docking, and biological activity of polyfluoroalkyl dihydroazolo[5,1-c][1,2,4]triazines as selective carboxylesterase inhibitors

Multiple binding of D-tubocurarine to acetylcholinesterase

The high catalytic efficiency of enzymes under reaction conditions is one of the main goals in biocatalysis. Despite the dramatic progress in the development of more efficient biocatalysts by protein design, the search for natural enzymes with useful properties remains a promising strategy. The pyridoxal 5'-phosphate (PLP)-dependent transaminases represent a group of industrially important enzymes due to their ability to stereoselectively transfer amino groups between diverse substrates; however, the complex mechanism of substrate recognition and conversion makes the design of transaminases a challenging task. Here we report a detailed structural and kinetic study of thermostable transaminase from the bacterium Thermobaculum terrenum (TaTT) using the methods of enzyme kinetics, X-ray crystallography and molecular modeling. TaTT can convert L-branched-chain and L-aromatic amino acids as well as (R)-(+)-1-phenylethylamine at a high rate and with high enantioselectivity. The structures of TaTT in complex with the cofactor pyridoxal 5′-phosphate covalently bound to enzyme and in complex with its reduced form, pyridoxamine 5′-phosphate, were determined at resolutions of 2.19 A and 1.5 A, and deposited in the Protein Data Bank as entries 6GKR and 6Q8E, respectively. TaTT is a fold type IV PLP-dependent enzyme. In terms of structural similarity, the enzyme is close to known branched-chain amino acid aminotransferases, but differences in characteristic sequence motifs in the active site were observed in TaTT compared to canonical branched-chain amino acid aminotransferases, which can explain the improved binding of aromatic amino acids and (R)-(+)-1-phenylethylamine. This study has shown for the first time that high substrate specificity towards both various l -amino acids and (R)-primary amines can be implemented within one pyridoxal 5′-phosphate-dependent active site of fold type IV. These results complement our knowledge of the catalytic diversity of transaminases and indicate the need for further biochemical and bioinformatic studies to understand the sequence-structure-function relationship in these enzymes.

Methods of Enzyme Kinetics