scholarly journals Steady-state parameters of an enzyme from n.m.r. spin transfer with thermal variation

1987 ◽  
Vol 244 (1) ◽  
pp. 247-248 ◽  
Author(s):  
P W Kuchel
Keyword(s):  
Steady State ◽  
Rate Constants ◽  
Spin Exchange ◽  
Enzyme System ◽  
Radioactive Tracer ◽  
Solute Concentration ◽  
Spin Transfer ◽  
Thermal Variation ◽  
The Individual ◽  
Individual Rate

N.m.r. spin-exchange analysis of enzymic reactions at chemical equilibrium is akin to radioactive-tracer-exchange analysis; unidirectional flux rates are obtained for the overall reaction. These data, by themselves, are not sufficient to define the values of all the individual rate constants or steady-state parameters. However, it is shown that, by measuring the dependence of the exchange rate constants on solute concentration and temperature, the individual rate constants, and hence the steady-state parameters, can be obtained for a simple enzyme system.

scholarly journals Isomerization of an enzyme-coenzyme complex in yeast alcohol dehydrogenase-catalysed reactions

2003 ◽  
Vol 68 (2) ◽  
pp. 77-84 ◽  
Author(s):  
Vladimir Leskovac ◽  
Svetlana Trivic ◽  
Draginja Pericin
Keyword(s):  
Alcohol Dehydrogenase ◽  
Rate Constants ◽  
Equilibrium Constants ◽  
Kinetic Mechanism ◽  
Yeast Alcohol Dehydrogenase ◽  
Catalyzed Reactions ◽  
The Individual ◽  
Individual Rate ◽  
Catalyzed Oxidation

In this work, all the rate constants in the kinetic mechanism of the yeast alcohol dehydrogenase-catalyzed oxidation of ethanol by NAD+, at pH 7.0, 25 ?C, have been estimated. The determination of the individual rate constants was achieved by fitting the reaction progress curves to the experimental data, using the procedures of the FITSIM and KINSIM software package of Carl Frieden. This work is the first report in the literature showing the internal equilibrium constants for the isomerization of the enzyme-NAD+ complex in yeast alcohol dehydrogenase-catalyzed reactions.

scholarly journals The linkage between binding of the C-terminal domain of hirudin and amidase activity in human α-thrombin

1993 ◽  
Vol 289 (2) ◽  
pp. 475-480 ◽  
Author(s):  
R de Cristofaro ◽  
B Rocca ◽  
B Bizzi ◽  
R Landolfi
Keyword(s):  
Rate Constants ◽  
Binding Constant ◽  
Amidase Activity ◽  
Equilibrium Binding ◽  
Viscosity Effects ◽  
The Individual ◽  
Individual Rate ◽  
Hydrolysis Of ◽  
Equilibrium Binding Constant

A method derived from the analysis of viscosity effects on the hydrolysis of the amide substrates D-phenylalanylpipecolyl-arginine-p-nitroaniline, tosylglycylprolylarginine-p-nitroanaline and cyclohexylglycylalanylarginine-p-nitroalanine by human alpha-thrombin was developed to dissect the Michaelis-Menten parameters Km and kcat into the individual rate constants of the binding, acylation and deacylation reactions. This method was used to analyse the effect of the C-terminal hirudin (residues 54-65) [hir-(54-65)] domain on the binding and hydrolysis of the three substrates. The results showed that the C-terminal hir-(54-65) fragment affects only the acylation rate, which is increased approx. 1.2-fold for all the substrates. Analysis of the dependence of acylation rate constants on hirudin-fragment concentration, allowed the determination of the equilibrium binding constant of C-terminal hir-(54-65) (Kd approximately 0.7 microM). In addition this peptide was found to competitively inhibit thrombin-fibrinogen interaction with a Ki which is in excellent agreement with the equilibrium constant derived from viscosity experiments. These results demonstrate that binding of hir-(54-65) to the fibrinogen recognition site of thrombin does not affect the equilibrium binding of amide substrates, but induces only a small increase in the acylation rate of the hydrolysis reaction.

Acid-catalyzed hydrolysis of 2-methylene-1,3-dithiolane. 2. Remarkable effects of β substitution on reversibility of the carbon protonation

1986 ◽  
Vol 64 (6) ◽  
pp. 1116-1123 ◽  
Author(s):  
Tadashi Okuyama ◽  
Masayoshi Toyoda ◽  
Takayuki Fueno
Keyword(s):  
Aqueous Solution ◽  
Rate Constants ◽  
Isotope Exchange ◽  
Relative Stabilities ◽  
The Third ◽  
Initial Carbon ◽  
Acid Catalyzed ◽  
The Individual ◽  
Individual Rate ◽  
Hydrolysis Of

Hydrolyses of 2-ethylidene-(1b), 2-isopropylidene-(1c), and 2-benzylidene-1,3-dithiolane (1d) were kinetically investigated in aqueous solution. All the individual rate constants involved in this three-step reaction were evaluated. Initial carbon protonation is only partially reversible (k2/k−1 = 1.33, 0.68, and 1.02 for 1b, 1c, and 1d, respectively) at higher pH, while the protonation becomes completely reversible below pH 2 where the third step is rate determining. Complete H–D isotope exchange at the β-carbon of 1b and 1d was observed in deuterium media before appreciable hydrolysis took place. It was demonstrated that reversion from the tetrahedral intermediate 3 to 1 occurs extensively during the reaction in the latter acidity range. Relative stabilities and reactivities of the olefinic substrates 1 are discussed.

scholarly journals Temperature response of Rubisco kinetics in Arabidopsis thaliana: thermal breakpoints and implications for reaction mechanisms

2018 ◽  
Author(s):  
Ryan A. Boyd ◽  
Amanda P. Cavanagh ◽  
David S. Kubien ◽  
Asaph B. Cousins
Keyword(s):  
Arabidopsis Thaliana ◽  
Reaction Mechanism ◽  
Kinetic Parameters ◽  
Rate Constants ◽  
Reaction Mechanisms ◽  
Temperature Response ◽  
Concentration Changes ◽  
The Individual ◽  
Temperature Responses ◽  
Individual Rate

ABSTRACTOptimization of Rubisco kinetics could improve photosynthetic efficiency, ultimatly resulting in increased crop yield. However, imprecise knowledge of the reaction mechanism and the individual rate constants limit our ability to optimize the enzyme. Membrane inlet mass spectrometery (MIMS) may offer benefits over traditional methods for determining individual rate constants of the Rubisco reaction mechanism, as it can directly monitor concentration changes in CO2, O2, and their isotopologs during assays. However, a direct comparsion of MIMS to the traditional Radiolabel method of determining Rubisco kinetic parameters has not been made. Here, the temperature responses of Rubisco kinetic parameters from Arabidopsis thaliana were measured using the Radiolabel and MIMS methods. The two methods provided comparable parameters above 25 °C, but temperature responses deviated at low temperature as MIMS derived catalytic rates of carboxylation, oxygenation, and CO2/O2 specificity showed thermal breakpoints. Here we discuss the variability and uncertainty surrounding breakpoints in the Rubisco temperature response and relavance of individual rate constants of the reaction mechanisms to potential breakpoints.

scholarly journals Comparison of Eight Methods for the Estimation of the Image-Derived Input Function in Dynamic [18F]-FDG PET Human Brain Studies

2009 ◽  
Vol 29 (11) ◽  
pp. 1825-1835 ◽  
Author(s):  
Paolo Zanotti-Fregonara ◽  
El Mostafa Fadaili ◽  
Renaud Maroy ◽  
Claude Comtat ◽  
Antoine Souloumiac ◽  
...  
Keyword(s):  
Blood Sample ◽  
Rate Constants ◽  
Fdg Pet ◽  
Input Function ◽  
Blood Samples ◽  
Arterial Blood ◽  
Positron Emission ◽  
Phantom Studies ◽  
The Individual ◽  
Individual Rate

The aim of this study was to compare eight methods for the estimation of the image-derived input function (IDIF) in [18F]-FDG positron emission tomography (PET) dynamic brain studies. The methods were tested on two digital phantoms and on four healthy volunteers. Image-derived input functions obtained with each method were compared with the reference input functions, that is, the activity in the carotid labels of the phantoms and arterial blood samples for the volunteers, in terms of visual inspection, areas under the curve, cerebral metabolic rates of glucose (CMRglc), and individual rate constants. Blood-sample-free methods provided less reliable results as compared with those obtained using the methods that require the use of blood samples. For some of the blood-sample-free methods, CMRglc estimations considerably improved when the IDIF was calibrated with a single blood sample. Only one of the methods tested in this study, and only in phantom studies, allowed a reliable calculation of the individual rate constants. For the estimation of CMRglc values using an IDIF in [18F]-FDG PET brain studies, a reliable absolute blood-sample-free procedure is not available yet.

Mutarotation of D(+)-glucose. II. Volumes of activation for the uncatalysed and copper(II)-catalysed rates

1983 ◽  
Vol 36 (2) ◽  
pp. 279 ◽  
Author(s):  
CJ O'Conner ◽  
AL Odell ◽  
AAT Bailey
Keyword(s):  
High Pressure ◽  
Reaction Mechanism ◽  
Equilibrium Constant ◽  
Rate Constants ◽  
Reaction Mechanisms ◽  
Sodium Perchlorate ◽  
Continuous Series ◽  
Aqueous Sodium ◽  
The Individual ◽  
Individual Rate

The effect of high pressure on the rate of mutarotation of α-and β-D(+)-glucose in aqueous sodium perchlorate solutions has been evaluated. The observed rate constant kΨ = kα + kβ increases with pressure while the equilibrium constant Keq = kα /kβ is nearly unchanged from the 1.7 found at 1 bar. From the experimental results, the individual rate constants kα and kβ and the corresponding activation volumes ∆V‡α and ∆V‡β can be evaluated. The values obtained, -10.7 � 0.1 and -10.0 � 0.1 cm3 mol-1, are discussed in relation to the reaction mechanism. In the presence of Cu(ClO4)2 the values of ∆V: form a continuous series, dependent upon the pH and the concentration of copper(II) catalyst, up to + 1.3 cm3 mol-1. The activation volumes for mutarotation by monomeric and dimeric copper(II) catalysts, ∆V‡(Cu2+) and ∆V‡ {CU((OH)2Cu)2+}, are -1.2 � 0.2 and + 3.6 � 0.2 cm3 mol-1 respectively. Reaction mechanisms are discussed to account for these values of ∆V‡.

Estimation of the individual rate constants for the steps in the thermal decomposition of ethane

1963 ◽  
Vol 271 (1344) ◽  
pp. 34-43 ◽  
Keyword(s):  
Thermal Decomposition ◽  
Rate Constants ◽  
General Pattern ◽  
Reaction Scheme ◽  
Methyl Radicals ◽  
Methyl Radical ◽  
Pattern Of Variation ◽  
The Individual ◽  
Limiting Case ◽  
Individual Rate

A reaction scheme analogous to that used for the higher paraffins (Blackmore & Hinshelwood 1962 b ) is tested for its applicability to the thermal decomposition of ethane and its inhibition by nitric oxide. Ethane constitutes a limiting case for the paraffin series, in that Hatomsre ­place methyl radicals as chain-carrying species. The individual rate constants of the reaction steps are estimated on the basis of this scheme, and will all conform satisfactorily to the general pattern of variation in the homologous series, allowance being made for the discontinuous increase expected when an H atom appears instead of a methyl radical.

A New Method for Determining Individual Rate Constants for Specific Non-chromophoric Substrates of Proteolytic Enzymes

1975 ◽  
Vol 53 (4) ◽  
pp. 564-571
Author(s):  
Lewis J. Brubacher
Keyword(s):  
Steady State ◽  
Rate Constants ◽  
Proteolytic Enzymes ◽  
Enzyme Mechanism ◽  
Steady State Kinetics ◽  
Steady State Rate ◽  
State Rate ◽  
Individual Rate ◽  
Kinetics Of ◽  
Hydrolysis Of

Equations are developed for the pre-steady state kinetics of the proteolytic enzyme-catalyzed hydrolysis of a substrate A in the presence of a monitoring substrate (or covalent inhibitor) S of known properties. A two-intermediate acyl–enzyme mechanism is assumed in which the first intermediate is in instantaneous equilibrium with enzyme and substrate. The appearance of the first product of substrate S is characterized by two relaxation rate constants. From these constants it is possible to determine the dissociation constant and the acylation and deacylation rate constants of substrate A. Criteria are also developed for using the steady state rate parameters of A to establish conditions for which the slower relaxation process is equivalent to the deacylation rate constant of A. The technique of premixing enzyme with substrate A has certain advantages in this approach.

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