PH-activity curves for enzyme-catalysed reactions in which the hydron is a product or reactant.
Ontology highlight
ABSTRACT:
SUBMITTER: Dixon HB
PROVIDER: S-EPMC1148580 | biostudies-other | 1987 Dec
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Pericyclic reactions-which proceed in a concerted fashion through a cyclic transition state-are among the most powerful synthetic transformations used to make multiple regioselective and stereoselective carbon-carbon bonds. They have been widely applied to the synthesis of biologically active complex natural products containing contiguous stereogenic carbon centres. Despite the prominence of pericyclic reactions in total synthesis, only three naturally existing enzymatic examples (the intramolecular Diels-Alder reaction, and the Cope and the Claisen rearrangements) have been characterized. Here we report a versatile S-adenosyl-l-methionine (SAM)-dependent enzyme, LepI, that can catalyse stereoselective dehydration followed by three pericyclic transformations: intramolecular Diels-Alder and hetero-Diels-Alder reactions via a single ambimodal transition state, and a retro-Claisen rearrangement. Together, these transformations lead to the formation of the dihydropyran core of the fungal natural product, leporin. Combined in vitro enzymatic characterization and computational studies provide insight into how LepI regulates these bifurcating biosynthetic reaction pathways by using SAM as the cofactor. These pathways converge to the desired biosynthetic end product via the (SAM-dependent) retro-Claisen rearrangement catalysed by LepI. We expect that more pericyclic biosynthetic enzymatic transformations remain to be discovered in naturally occurring enzyme 'toolboxes'. The new role of the versatile cofactor SAM is likely to be found in other examples of enzyme catalysis.
| S-EPMC5679075 | biostudies-literature
Project description:1. Several methods of analysing progress curves of enzyme-catalysed reactions are discussed briefly in relation to their usefulness in a situation where a reaction product has a K(i) much lower than the K(m) for the substrate 2. A comparison is made of different methods of estimating initial rates in this situation. 3. The use of a computer curve-fitting routine capable of handling functions of more than one variable for the extraction of kinetic parameters from progress curves is described. 4. This method and that of fitting time as a polynomial in product concentration are applied to progress curves for the soluble mitochondrial adenosine triphosphatase and the results are compared with values obtained by more conventional methods.
| S-EPMC1165854 | biostudies-other
Project description:Monitoring the kinetics and conformational dynamics of single enzymes is crucial to better understand their biological functions because these motions and structural dynamics are usually unsynchronized among the molecules. However, detecting the enzyme-reactant interactions and associated conformational changes of the enzyme on a single-molecule basis remains as a challenge to established optical techniques because of the commonly required labeling of the reactants or the enzyme itself. The labeling process is usually nontrivial, and the labels themselves might skew the physical properties of the enzyme. We demonstrate an optical, label-free method capable of observing enzymatic interactions and associated conformational changes on a single-molecule level. We monitor polymerase/DNA interactions via the strong near-field enhancement provided by plasmonic nanorods resonantly coupled to whispering gallery modes in microcavities. Specifically, we use two different recognition schemes: one in which the kinetics of polymerase/DNA interactions are probed in the vicinity of DNA-functionalized nanorods, and the other in which these interactions are probed via the magnitude of conformational changes in the polymerase molecules immobilized on nanorods. In both approaches, we find that low and high polymerase activities can be clearly discerned through their characteristic signal amplitude and signal length distributions. Furthermore, the thermodynamic study of the monitored interactions suggests the occurrence of DNA polymerization. This work constitutes a proof-of-concept study of enzymatic activities using plasmonically enhanced microcavities and establishes an alternative and label-free method capable of investigating structural changes in single molecules.
| S-EPMC5371424 | biostudies-literature
Project description:A simple case is considered in which the rate of a two-step reaction depends on pH because the intermediate formed in the first step has to gain (or lose) a proton before it can react in the second step, and in which the rate-determining step therefore changes with pH. The curves of reaction rate against pH are shown to be symmetrical, and the sharpest peak possible has a width at half its height of 1.53pH units, i.e. of 2log(3+2 radical2). Any particular curve for this situation proves to be identical with a curve that could be generated for the pH-dependence of a single-step reaction in which the rate is proportional to the concentration of a particular ionic form of a reactant. Curves for the latter situation, however, can have forms impossible for the former case in which the rate-determining step changes, but only if the protonations that activate and deactivate the reactant are co-operative. The peak can then become even sharper, and its width at half its height can fall to 1.14pH units, i.e. to 2log(2+ radical3).
| S-EPMC1177448 | biostudies-other
Project description:Heat capacity changes are emerging as essential for explaining the temperature dependence of enzyme-catalysed reaction rates. This has important implications for enzyme kinetics, thermoadaptation and evolution, but the physical basis of these heat capacity changes is unknown. Here we show by a combination of experiment and simulation, for two quite distinct enzymes (dimeric ketosteroid isomerase and monomeric alpha-glucosidase), that the activation heat capacity change for the catalysed reaction can be predicted through atomistic molecular dynamics simulations. The simulations reveal subtle and surprising underlying dynamical changes: tightening of loops around the active site is observed, along with changes in energetic fluctuations across the whole enzyme including important contributions from oligomeric neighbours and domains distal to the active site. This has general implications for understanding enzyme catalysis and demonstrating a direct connection between functionally important microscopic dynamics and macroscopically measurable quantities.
| S-EPMC5862990 | biostudies-literature
Project description:Most methods for studying the kinetic properties of an enzyme involve the determination of initial velocities. When the reaction progress curve shows significant curvature due to depletion of the substrate, accumulation of inhibitory products or instability of the enzyme, estimation of the initial velocity is a subjective and inexact process. Two methods have been suggested [Cornish-Bowden (1975) Biochem. J. 144, 305-312; Boeker (1982) Biochem J. 203, 117-123] that attempt to eliminate this subjective element. The present study offers a third alternative, which is based on fitting a reparameterized form of the integrated Michaelis-Menten equation to the progress curves by non-linear regression. This method yields estimates and standard errors of the initial velocity and of the time to reach 50% reaction. No prior knowledge of the apparent product concentration at zero time or infinite time is required, since both of these quantities are also estimated from the data. It is shown that this method yields reliable estimates of the initial velocity under a wide range of circumstances, including those where the two previously published methods perform poorly.
| S-EPMC1144952 | biostudies-other
Project description:1. The pH-dependence of the pepsin-catalysed hydrolysis of three peptide substrates was studied by using a method for the continuous monitoring of the formation of ninhydrin-positive products. 2. Two peptide acid substrates, N-acetyl-l-phenylalanyl-l-phenylalanine and N-acetyl-l-phenylalanyl-l-phenylalanyl-glycine, show apparent pK(a) values of 1.1 and 3.5 in the plots of k(0)/K(m) versus pH. By contrast a neutral substrate, N-acetyl-l-phenylalanyl-l-phenylalanine amide, shows apparent pK(a) values of 1.0 and 4.7. 3. Together with the data of the preceding paper (Knowles, Sharp & Greenwell, 1969), these results are taken to indicate that the rate of pepsin-catalysed hydrolysis is controlled by the ionization of two groups, which on the free enzyme have apparent pK(a) values of 1.0 and 4.7. It is apparent that the anions of peptide acid substrates are not perceptibly bound to the enzyme, resulting in apparent pK(a) values of 3.5 for the dependence of k(0)/K(m) for these materials.
| S-EPMC1184642 | biostudies-other
Project description:The computer analysis of progress curves for enzyme-catalysed reactions involves a series of mathematical and computational tasks. The three most daunting of these are the derivation of an integrated rate equation, solving this equation so that the amount of product formed by the reaction at any time can be calculated, and incorporating this solution into a non-linear-regression computer program. This paper describes the basis of a computer program that greatly simplifies the problem. The proposed mechanism is specified in the familiar kinetic constant form, which is automatically translated into a program capable of fitting this mechanism to a series of experimental progress curves. The approach is illustrated for a reversible reaction with one substrate and one product, and tested with some data obtained for the fumarase reaction. A copy of the program has been deposited as Supplementary Publication SUP 50148 (13 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1989) 257, 5.
| S-EPMC1138375 | biostudies-other
Project description:It is now widely accepted that enzyme-catalysed C-H bond breakage occurs by quantum mechanical tunnelling. This paradigm shift in the conceptual framework for these reactions away from semi-classical transition state theory (TST, i.e. including zero-point energy, but with no tunnelling correction) has been driven over the recent years by experimental studies of the temperature dependence of kinetic isotope effects (KIEs) for these reactions in a range of enzymes, including the tryptophan tryptophylquinone-dependent enzymes such as methylamine dehydrogenase and aromatic amine dehydrogenase, and the flavoenzymes such as morphinone reductase and pentaerythritol tetranitrate reductase, which produced observations that are also inconsistent with the simple Bell-correction model of tunnelling. However, these data-especially, the strong temperature dependence of reaction rates and the variable temperature dependence of KIEs-are consistent with other tunnelling models (termed full tunnelling models), in which protein and/or substrate fluctuations generate a configuration compatible with tunnelling. These models accommodate substrate/protein (environment) fluctuations required to attain a configuration with degenerate nuclear quantum states and, when necessary, motion required to increase the probability of tunnelling in these states. Furthermore, tunnelling mechanisms in enzymes are supported by atomistic computational studies performed within the framework of modern TST, which incorporates quantum nuclear effects.
| S-EPMC1647315 | biostudies-literature
Project description:For enzyme-catalysed biotransformations, continuous in situ detection methods minimise the need for sample manipulation, ultimately leading to more accurate real-time kinetic determinations of substrate(s) and product(s). We have established for the first time an on-line, real-time quantitative approach to monitor simultaneously multiple biotransformations based on UV resonance Raman (UVRR) spectroscopy. To exemplify the generality and versatility of this approach, multiple substrates and enzyme systems were used involving nitrile hydratase (NHase) and xanthine oxidase (XO), both of which are of industrial and biological significance, and incorporate multistep enzymatic conversions. Multivariate data analysis of the UVRR spectra, involving multivariate curve resolution-alternating least squares (MCR-ALS), was employed to effect absolute quantification of substrate(s) and product(s); repeated benchmarking of UVRR combined with MCR-ALS by HPLC confirmed excellent reproducibility.
| S-EPMC5488198 | biostudies-literature