Kinetics Of Enzyme-catalyzed Reactions Research Articles

Leveraging Isothermal Titration Calorimetry to Obtain Thermodynamic Insights into the Binding Behavior and Formation of Metal-Organic Frameworks.

Isothermal titration calorimetry (ITC) is a technique which directly measures the thermodynamic parameters of binding events. Although historically it has been used to investigate interactions in biological macromolecules and the kinetics of enzyme-catalyzed reactions, ITC has also been demonstrated to provide relevant thermodynamic information about interactions in synthetic systems, such as those in metal-organic frameworks (MOFs). MOFs are a family of crystalline porous materials that have been widely studied as supports for molecules ranging from gases to biomolecules through physisorption and chemisorption. Herein, we offer a perspective on the current applications of ITC in MOFs, including the mechanism of small molecule adsorption and the formation of MOF-based composite materials through noncovalent interactions. Experimental considerations specific to running ITC experiments in MOF systems are reviewed on the basis of existing reports. We conclude by discussing underexplored, but promising, MOF-related research directions which could be elucidated by ITC.

Distinct kinetic mechanisms of H3K4 methylation catalyzed by MLL3 and MLL4 core complexes

The methyltransferases MLL3 and MLL4 primarily catalyze the monomethylation of histone H3 lysine 4 (H3K4) on enhancers to regulate cell-type-specific gene expression and cell fate transition. MLL3 and MLL4 share almost identical binding partners and biochemical activities, but perform specific and nonredundant functions. The features and functions that distinguish MLL3 and MLL4 remain elusive. Here, we characterize the kinetic mechanisms of MLL3 and MLL4 ternary complexes containing the catalytic SET domain from MLL3 or MLL4 (MLL3SET or MLL4SET), the SPRY domain of ASH2L (ASH2LSPRY), and a short fragment of RBBP5 (RBBP5AS-ABM) to search for possible explanations. Steady-state kinetic analyses and inhibition studies reveal that the MLL3 complex catalyzes methylation in a random sequential bi–bi mechanism. In contrast, the MLL4 complex adopts an ordered sequential bi–bi mechanism, in which the cofactor S-adenosylmethionine (AdoMet) binds to the enzyme prior to the H3 peptide, and the methylated H3 peptide dissociates from the enzyme before S-adenosylhomocysteine (AdoHcy) detaches after methylation. Substrate-binding assays using fluorescence polarization (FP) confirm that AdoMet binding is a prerequisite for H3 binding for the MLL4 complex but not for the MLL3 complex. Molecular dynamic simulations reveal that the binding of AdoMet exclusively induces conformational constraints on the AdoMet-binding groove and the H3 substrate-binding pocket of MLL4, therefore stabilizing a specific active conformation to ease entry of the substrate H3. The distinct kinetic mechanisms and conformational plasticities provide important insights into the differential functions of MLL3 and MLL4 and may also guide the development of selective inhibitors targeting MLL3 or MLL4.

Inverse counting statistics for stochastic and open quantum systems: the characteristic polynomial approach

We consider stochastic and open quantum systems with a finite number of states, where a stochastic transition between two specific states is monitored by a detector. The long-time counting statistics of the observed realizations of the transition, parametrized by cumulants, is the only available information about the system. We present an analytical method for reconstructing generators of the time evolution of the system compatible with the observations. The practicality of the reconstruction method is demonstrated by the examples of a laser-driven atom and the kinetics of enzyme-catalyzed reactions. Moreover, we propose cumulant-based criteria for testing the non-classicality and non-Markovianity of the time evolution, and lower bounds for the system dimension. Our analytical results rely on the close connection between the cumulants of the counting statistics and the characteristic polynomial of the generator, which takes the role of a cumulant generating function.

Levitated Drop Microreactors for Biochemical Kinetics

Ultrasonically-levitated drops have been widely studied for materials processing and for sample preparation for chemical analysis. We report on the development of such drops for study of kinetics of enzyme-catalyzed reactions and other chemical processes. We review how to simply and reliably levitate drops, discuss why such drops are desirable for studying biochemical reactions, especially those generating or consuming free radicals, and report progress towards routine kinetics measurements in microliter drops.

EFFECTS OF GREEN TEA EXTRACT ON “GOLDEN DELICIOUS” APPLE POLYPHENOLOXIDASE AND ITS BROWNING

ABSTRACT The inhibitory effect of green tea extract on apple polyphenol oxidase (PPO) and on apple browning was investigated. PPO has been isolated from apples (cv. Golden Delicious), and extraction with polyvinylpolypyrrolidone and Triton X100 yielded a 6.45-fold increase in the specific enzyme activity. The optimum pH and temperature of the enzyme was found as 5.5 and 25C, respectively. The kinetics of enzyme-catalyzed reactions followed the Michaelis-Menten model, and the PPO exhibited a Km value of 4.76 mM. Apple PPO activity was inhibited by green tea extract, and the inhibitory effect toward apple PPO increased with increasing extract concentration. Addition of 30 mg/mL green tea extract resulted in approximately 42% inhibition on apple PPO activity. To support the inhibition behavior of PPO, apple browning was also investigated by immersing apple slices into green tea extract, and it was revealed that the browning of apple slices was effectively retarded by the green tea extract. PRACTICAL APPLICATIONS The quality of fruits, especially apples, depends on controlling the enzymatic browning that affects its organoleptic and nutritional qualities. Addition of chemicals, pH adjustment, refrigeration, thermal treatments and exclusion of oxygen are among the most effective methods for the inhibition of polyphenol oxidases (PPO). Consumer demand for minimally processed and fresh-like food products has undergone a continuous rise in recent years. Several authors have stated the necessity to evaluate the inhibitory effect of natural and nontoxic compounds on PPO activity and its potential applicability to the food industry. Experiments showed that green tea has an inhibitory effect on the activity of apple PPO and its browning. Our results suggest that green tea extract could potentially be used as a natural inhibitor of PPO, and its utilization is possible due to its wide occurrence, low cost and nontoxicity, but further feasibility studies are needed to increase its effect as an anti-browning agent in its application in the food industry.

Effect of partitioning equilibria on the activity of β-galactosidase in heterogeneous media

We had demonstrated that membrane adsorption or penetration differentially modulated β-Galactosidase (β-Gal) activity against soluble substrates (Coll. and Surf., 24, 21, 2002). In a heterogeneous media, not only the enzyme but also the rest of the chemical species taking part in a chemical reaction would eventually interact with the available surfaces. The aim of the present work was to investigate if, in addition to changes in the intrinsic mechanism of the reaction at the lipid–water interface, the kinetics of enzyme-catalyzed reactions could be significantly affected by the partitioning of the substrate ( ortho-nitro-phenyl galactopyranoside (ONPG)), the product ( ortho-nitro-phenol (ONP)) and the enzyme ( E. coli β-Gal) towards the membrane. Multilamellar vesicles of sPC were used as model membranes. Membrane–water partition coefficients ( P m/w) were determined according to the theory and methodology developed previously (J. Neurosci. Meth. 36, 203, 1991). The values of P m/w obtained ( P ONPG = 0, P ONP = 50 and P β-Gal = 118) were applied to a two-compartment model, which assumed a free access of the substrate to the enzyme and a nucleophile-like activatory effect exerted, within the membrane compartment, by the lipid–water interface. This model: (i) reproduced the lipid concentration-dependence we had observed previously in V max, (ii) predicted the values of k 4 = 3.54 × 10 7 s − 1 and the extinction coefficient of the aglycone in the membrane phase, 4012 M − 1 cm − 1 , with p < 0.0001 and p < 0.02, respectively, as well as for P β-Gal = 117 (which was poor ( p = 0.6716) but gave a numerical value within the same order of magnitude that the experimental value) and (iii) emphasized the importance of the more efficient reaction mechanism in the membrane phase compared with that in the aqueous phase (k 4 ≫ k 3).

A thermogravimetric method for studying the kinetics of enzyme catalysed reactions that produce a volatile co-product

A general method, using a thermogravimetric balance, has been developed for determining the rate of enzyme reactions that involve loss of a volatile co-product in the presence of an involatile product, the method requiring only small amounts of both substrate and enzyme. Following our interest in enzyme mediated polyester formation, the method was developed using the Candida antarctica lipase B catalysed reaction of various diols with adipic acid or monoethyl adipate (MEAA) to form polyesters and water or ethanol, respectively. The reactions were shown to be first order in enzyme concentration. 1,4-Butanediol was found to be too volatile for accurate rate determination, but the volatilities of 1,6-hexanediol and polytetramethylene diol (PTMEG 650) were sufficiently low for reaction rates to be determined below 80 °C for 1,6-hexanediol and 140 °C with PTMEG 650. Although Candida antarctica lipase B is a thermozyme it does show significant degradation of reaction rate above 130 °C. Arrhenius parameters for the reaction of 1,6-hexanediol with adipic acid and MEAA confirm that the enzyme has a distinct preference for reaction with ester rather than carboxylic acid groups.

27] Pressure effects on activity and stability of hyperthermophilic enzymes

Publisher Summary Along with high temperatures, high pressures [ca. 200-300 atm (1 atm = 101.29 kPa)] are a salient feature of deep-sea hydrothermal vents, one of the most violent environments on earth and a rich source of hyperthermophilic organisms. Thermophilic bacteria have also been isolated from the cores of oil and sulphur wells, and the existence of microbial life in other environments of high temperature and pressure appears likely. Although much work has been devoted to the investigation of high-temperature adaptation of enzymes from thermophiles and hyperthermophiles, relatively few studies have focused on the adaptation of these enzymes to high pressure. However, high pressure may play an important role in determining the functional properties of enzymes from organisms conditioned to high temperature-pressure environments. High pressure can be a very useful tool for studying the structure and function of proteins, as evidenced by high-pressure studies of protein folding/unfolding pathways, the kinetics of enzyme-catalyzed reactions, and enzymatic reaction mechanisms in nonaqueous media. This chapter describes some of the special techniques and equipment used for high-pressure activity and stability studies of hyperthermophilic enzymes in the authors' laboratory, along with results obtained from investigations of hyperthermophilic enzymes.

Theory and Simulation of the Influence of Diffusion in Enzyme-Catalyzed Reactions

The Michaelis−Menten equation for the kinetics of a simple enzyme-catalyzed reaction is based on the assumption that the two steps of the reaction, (i) reversible formation of the enzyme−substrate complex (ES) by diffusional encounter and (ii) irreversible conversion of the substrate in ES to product, are both described by ordinary rate equations. It is well-known that the rate coefficient, k(t), for enzyme−substrate binding is time dependent due to the influence of diffusion. Will the influence of diffusion lead to non-Michaelis−Menten kinetics? To address this question, three theoretical approaches to account for the influence of diffusion on the kinetics of enzyme-catalyzed reactions are discussed and tested on a model system. It is found that the restriction on the site for enzyme−substrate binding makes the time dependence of k(t) sufficiently weak so that deviation from the Michaelis−Menten equation is unlikely to be observed. Within the range of parameters that is of practical interest, the three t...

A Tyrosine Kinase Assay Using Reverse-Phase High-Performance Liquid Chromatography

Reverse-phase HPLC can be used as a very precise and accurate routine assay for peptide phosphorylation by protein kinases that has advantages over other methods. In particular, peptides with native amino acid sequences can be used without the need for radioisotopes. However, reaction conditions that are employed can often present difficulties in recovery and quantitation of phospho- and apo-peptides. Two general problems were encountered: First, variation in the retention times of peptides and an increasing width of the injection front which can interfere with quantitation both resulted from repeated sample injections. These were caused mostly by the presence of carrier bovine serum albumin used to reduce loss of peptides during the reaction and by high concentrations of ATP used to study the kinetics of enzyme catalyzed reactions. These problems were solved by regular washing of the reverse-phase column, thus allowing a broad range of peptide and ATP concentrations to be used. Second, the stability of peptides used in the assay was affected by dithiothreitol in combination with manganese. The former is a common reagent of kinase purifications and the latter is often the metal cofactor used in kinase reactions. Minimizing the concentration of dithiothreitol or using magnesium resolved these difficulties. Consideration of these factors is therefore important when using reverse-phase HPLC to monitor peptide phosphorylation in protein tyrosine kinase assays.

Solvation effects upon the thermodynamic substrate activity; correlation with the kinetics of enzyme catalyzed reactions. II. More complex interactions of alpha-chymotrypsin with dioxane and acetone which are also competitive inhibitors

It is shown that the effects of the addition of various amounts of dioxane and acetone (solvent modifiers) upon the α-chymotrypsin-catalyzed hydrolysis of methylhippurate can be explained in terms of three factors. (A) The effects of the above modifiers on the chemical potential of the substrate. (B) The solvent modifiers dioxane and acetone also act as classical competitive inhibitors. The means of sorting out these contributions is presented. (C) The alterations of the chemical potentials or for free energies of the enzyme, enzyme-substrate complex, and/or other intermediates by the added modifiers appear to cancel out with the substrate used here.

Solvation effects upon the thermodynamic substrate activity; correlation with the kinetics of enzyme catalyzed reactions. I. Effects of added reagents such as methanol upon alpha-chymotrypsin

Solvents, detergents, etc., have often been added to the medium to study the kinetics of enzyme action and for binding studies. They have been employed for diverse reasons such as solubilization of substrates or to stabilize an enzyme that was originally membrane bound. Thermodynamic considerations dictate that any added substance, such as methanol, which is present in significant quantity must affect the thermodynamic activities of the enzyme, enzyme-substrate complex, substrate and any other intermediates although cancellation effects may occur in this regard. The influence upon substrate activities is the only one that is easily experimentally accessible. These effects are shown, from the data of Bernard and Laidler, to be large in the case of the α-chymotrypsin catalysed hydrolysis of methylhydrocinnamate. The variation of the Michaelis-Menten constant is quantitatively explainable in terms of the alteration of the thermodynamic activity of the substrate by methanol.

Enzyme kinetics revisited: a commentaryon “The kinetics of enzyme-catalyzed reactions with two or more substrates or products’ by W.W. Cleland Biochim. Biophys. Acta 67 (1963) 104 ff. with Biochim. Biophys. Acta 67 (1963) 173, 188 (summaries)

Enzyme kinetics revisited: a commentaryon “The kinetics of enzyme-catalyzed reactions with two or more substrates or products’ by W.W. Cleland Biochim. Biophys. Acta 67 (1963) 104 ff. with Biochim. Biophys. Acta 67 (1963) 173, 188 (summaries)

Malcolm Dixon, 18 April 1899 - 7 December 1985

Malcolm Dixon died in Cambridge, the university city in which he spent the whole of his life, on 7 December 1985, at the age of 86. He achieved international distinction for his work on physical biochemistry, notably the purification of enzymes and the kinetics of enzyme-catalysed reactions, and he will long be remembered in addition for the leading part he played in the introduction of the present systematic nomenclature of enzymes. When he began research in 1921, even the chemical nature of enzymes as proteins was far from settled. Through his research and teaching, and the publication of three outstanding books, he did much to put the study of enzymes on a firm quantitative basis. And when he died, this intensely shy and private man, who lived according to deeply held Christian beliefs, was mourned by a wide circle of friends and admirers.

The influence of temperature-induced phase changes on the kinetics of respiratory and other membrane-associated enzyme systems.

Temperature-mediated changes in the kinetics of enzyme catalysed reactions can be due to effects on a number of different parameters. If the change in temperature does not (a) inactivate the enzyme, (b) alter the affinity of the enzyme for the substrate, an activator or an inhibitor or (c) alter the pH function of the reaction components, the velocity of enzyme catalysed reactions increases with increasing temperature. The relationship between the velocity of reaction and temperature can be expressed either as the activation energy (E) or the temperature coefficient (Q10). Both expressions can be derived from the empirical Arrhenius equation relating the velocity of reaction and temperature $$\frac{\alpha \ln k}{\alpha T}=\frac{E}{R{{T}^{2}}}$$ (1) where k is the reaction velocity constant, R the gas constant, T the absolute temperature and E a constant, subsequently called the activation energy (also written as A or μ.). Integration of equation (1) gives $$\ln \frac{{{k}_{2}}}{{{k}_{1}}}=\frac{E}{R}\left( \frac{1}{{{T}_{1}}}-\frac{1}{{{T}_{2}}} \right)$$ (2) from which it can be seen that the value for E can be obtained from the slope of the straight line when logk is plotted against 1/T $$E=2\cdot 303R\times slope$$ $$\therefore E=4\cdot 576\times slope \left( where R=1\cdot 987 cal/mole/{}^{\circ }K \right)$$

The kinetics of enzyme-catalyzed reactions with two or more substrates or products

The kinetics of enzyme-catalyzed reactions with two or more substrates or products

The kinetics of enzyme-catalyzed reactions with two or more substrates or products☆II. Inhibition: Nomenclature and theory

A nomenclature is proposed to describe different types of inhibitions of enzyme-catalyzed reactions, particularly for reactions with more than one substrate and product. The rate equations for such inhibitions are discussed, as are methods for distinguishing between the various types of inhibition and obtaining inhibition and kinetic constants from experimental data. Several examples are given of the type of information that can be obtained from inhibition studies.

The kinetics of enzyme-catalyzed reactions with two or more substrates or products

The kinetics of enzyme-catalyzed reactions with two or more substrates or products

The kinetics of enzyme-catalyzed reactions with two or more substrates or products

A general method for predicting initial velocity and dead end and product inhibition patterns by inspection of the mechanism is presented. This method is applicable to any non-random mechanism without alternate reaction sequences. Several examples of the application of the method are given.

The kinetics of enzyme-catalyzed reactions with two or more substrates or products. III. Prediction of initial velocity and inhibition patterns by inspection.

Abstract A general method for predicting initial velocity and dead end and product inhibition patterns by inspection of the mechanism is presented. This method is applicable to any non-random mechanism without alternate reaction sequences. Several examples of the application of the method are given.