scholarly journals Evolution of enzyme catalytic power. Characteristics of optimal catalysis evaluated for the simplest plausible kinetic model

1977 ◽  
Vol 163 (1) ◽  
pp. 111-116 ◽  
Author(s):  
Keith Brocklehurst
Keyword(s):  
Equilibrium Constant ◽  
Rate Constants ◽  
Catalytic Efficiency ◽  
Kinetic Mechanism ◽  
Enzyme Concentration ◽  
Forward Rate ◽  
Power Characteristics ◽  
Evolutionary Changes ◽  
Order Of Magnitude ◽  
Km Value

1. Evolutionary changes in the structure of an enzyme that provide an increase in its Km value are considered. Provided that Km increases as a result of increases in the forward rate constants of the catalysis relative to the reverse rate constants, the enzyme catalyses the conversion of a fixed concentration of its substrate more rapidly when its structure provides that Km>[S] than when Km<[S]. 2. Catalytic efficiency of enzymes is discussed in terms of the simplest plausible model, the Haldane [(1930) Enzymes, Longmans, London] reversible three-step model: [Formula: see text] The rate equation for the forward reaction of this model (formation of P) may be written in the simple form: [Formula: see text] Keq. is the equilibrium constant (=[P]eq./[S]eq.), and kcat.=V/[E]T, where [E]T is the total enzyme concentration. 3. To assess the effectiveness of an enzyme, it is necessary only to determine the extent to which the constraints of a particular kinetic mechanism permit v2 (v when Km»[S]) to approach vd (the diffusion-limited rate). 4. The value of the optimal rate of catalysis (vopt., the maximal value of v2) is dictated by the equilibrium constant for the reaction, Keq.; v2=vd/a, where [Formula: see text] when k+1 is assumed equal to k−3, and vopt.=vd/amin.. When Keq.≥1, it is necessary that k+2»k−1 for a to take its minimum value, amin.; when Keq.«1, it is necessary only that k+2»Keq.·k−1, i.e. a can equal amin. even if k+2<k−1. When Keq.»1, vopt.=vd; when Keq.=1, vopt.=vd/2, and when Keq.«1, vopt.=Keq.·vd. 5. The analysis, together with predicted effects of evolutionary pressure, suggests that in practice the rates of the fastest enzyme-catalysed freely reversible reactions might be expected to be lower than the value of k+1[E]T[S] by about an order of magnitude, particularly if Keq.<1. 6. The existing literature suggests that, in general, appropriate values of Km have evolved for the provision of high rates of catalysis but that many values of kcat. are not large enough to provide optimal rates of catalysis unless the value of k+1in vivo is lower than its value in free solution.

scholarly journals Phosphoglucoisomerase-catalysed interconversion of hexose phosphates. Kinetic study by 13C n.m.r. of the phosphoglucoisomerase reaction in 2H2O

1990 ◽  
Vol 265 (2) ◽  
pp. 519-524 ◽  
Author(s):  
R Willem ◽  
F Malaisse-Lagae ◽  
R Ottinger ◽  
W J Malaisse
Keyword(s):  
Proton Transfer ◽  
Kinetic Study ◽  
Rate Constant ◽  
Rate Constants ◽  
Strong Support ◽  
Enzyme Concentration ◽  
Intermolecular Proton Transfer ◽  
Order Of Magnitude ◽  
Single Exponential ◽  
Hexose Phosphates

The fate of D-[2-13C]glucose 6-phosphate exposed to phosphoglucoisomerase (glucose-6-phosphate isomerase, EC 5.3.1.9) in 2H2O was monitored by 13C-n.m.r. spectroscopy. The generation of the anomers of both D-[2-13C]fructose 6-phosphate and D-[2-13C,2-2H]glucose 6-phosphate followed a single-exponential pattern. The rate constant, which was proportional to the enzyme concentration, was about 14 times higher, however, in the former than in the latter case. The disappearance of D-[2-13C,2-1H]glucose 6-phosphate occurred in a bi-exponential manner, the rate constants for the fast and the slow processes being in fair agreement with those obtained for the generation of D-[2-13C]fructose 6-phosphate and D-[2-13C,2-2H]glucose 6-phosphate respectively. These findings indicate that the process of equilibration of D-[2-13C]glucose 6-phosphate and D-[2-13C]fructose 6-phosphate is at least one order of magnitude faster than the intermolecular proton transfer involving the deuterons from the solvent. Such a difference provides strong support to the view that the inverconversion of hexose phosphates in the reaction catalysed by phosphoglucoisomerase proceeds in two distinct steps, the second of which occurs according to two competing modalities with either an intramolecular or an intermolecular proton transfer.

The reaction of nickel(II) bis(diethyldithiocarbamate) with N,N,N',N'-tetraethyl thiuramdisulphide

1985 ◽  
Vol 50 (8) ◽  
pp. 1648-1660 ◽  
Author(s):  
Ernest Beinrohr ◽  
Andrej Staško ◽  
Ján Garaj
Keyword(s):  
Equilibrium Constant ◽  
Rate Constants

The oxidation of nickel(II) bis(diethyldithiocarbamate) (NiL2) by N,N,N',N'-tetraethyl thiuramdisulphide (tds) can be described by the equation 2 NiL2 + tds ⇄ 2 NiL3 (NiL3 = tris(diethyldithiocarbamate) nickel(III)). The equilibrium constant of the reaction depends on the polarity of the solvent (4.4 . 10-3 in toluene, 1.3 . 10-3 in chloroform, and 8 . 10-4 in acetone and methanol). The rate constants k1 and k-2 and the ratio k2/k-1 were found for the reaction steps NiL2 + tds ⇄ NiL3 + L. and NiL2 + L. ⇄ NiL3, where L. is the (C2H5)2NCS2. radical.

The Effect of p-Toluidine on the Two-Step Electroreduction of Zn(II) in Water-Methanol and Water-Dimethylformamide

1999 ◽  
Vol 64 (4) ◽  
pp. 585-594 ◽  
Author(s):  
Barbara Marczewska
Keyword(s):  
Electron Transfer ◽  
Binary Mixtures ◽  
Rate Constant ◽  
Rate Constants ◽  
Electrode Surface ◽  
Mercury Electrode ◽  
Forward Rate ◽  
Solvent Molecules ◽  
Activated Complex

The acceleration effect of p-toluidine on the electroreduction of Zn(II) on the mercury electrode surface in binary mixtures water-methanol and water-dimethylformamide is discussed. The obtained apparent and true forward rate constants of Zn(II) reduction indicate that the rate constant of the first electron transfer increases in the presence of p-toluidine. The acceleration effect may probably be accounted for by the concept of the formation on the mercury electrode an activated complex, presumably composed of p-toluidine and solvent molecules.

scholarly journals Reaction mechanism of the magnesium ion-dependent adenosine triphosphatase of frog muscle myosin and subfragment 1

1978 ◽  
Vol 171 (1) ◽  
pp. 165-175 ◽  
Author(s):  
M A Ferenczi ◽  
E Homsher ◽  
R M Simmons ◽  
D R Trentham
Keyword(s):  
Steady State ◽  
Rate Constants ◽  
Myosin Atpase ◽  
Forward Rate ◽  
Magnesium Ion ◽  
Skeletal Muscle Myosin ◽  
General Terms ◽  
Predominant Component ◽  
Main Components ◽  
Muscle Myosin

The Mg2+-dependent ATPase (adenosine 5′-triphosphatase) mechanism of myosin and subfragment 1 prepared from frog leg muscle was investigated by transient kinetic technique. The results show that in general terms the mechanism is similar to that of the rabbit skeletal-muscle myosin ATPase. During subfragment-1 ATPase activity at 0-5 degrees C pH 7.0 and I0.15, the predominant component of the steady-state intermediate is a subfragment-1-products complex (E.ADP.Pi). Binary subfragment-1-ATP (E.ATP) and subfragment-1-ADP (E.ADP) complexes are the other main components of the steady-state intermediate, the relative concentrations of the three components E.ATP, E.ADP.Pi and E.ADP being 5.5:92.5:2.0 respectively. The frog myosin ATPase mechanism is distinguished from that of the rabbit at 0-5 degrees C by the low steady-state concentrations of E.ATP and E.ADP relative to that of E.ADP.Pi and can be described by: E + ATP k' + 1 in equilibrium k' − 1 E.ATP k' + 2 in equilibrium k' − 2 E.ADP.Pi k' + 3 in equilibrium k' − 3 E.ADP + Pi k' + 4 in equilibrium k' − 4 E + ADP. In the above conditions successive forward rate constants have values: k' + 1, 1.1 × 10(5)M-1.S-1; k' + 2 greater than 5s-1; k' + 3, 0.011 s-1; k' + 4, 0.5 s-1; k'-1 is probably less than 0.006s-1. The observed second-order rate constants of the association of actin to subfragment 1 and of ATP-induced dissociation of the actin-subfragment-1 complex are 5.5 × 10(4) M-1.S-1 and 7.4 × 10(5) M-1.S-1 respectively at 2-5 degrees C and pH 7.0. The physiological implications of these results are discussed.

Deacylation of Fluorine-substituted trans-Cinnamoyl-α-chymotrypsins

1975 ◽  
Vol 53 (4) ◽  
pp. 513-518 ◽  
Author(s):  
J. T. Gerig ◽  
R. S. McLeod
Keyword(s):  
Rate Constants ◽  
Rate Effect ◽  
Ionization State ◽  
Hydrolysis Rates ◽  
Order Of Magnitude ◽  
Model Reactions

Deacylation of unsubstituted and of o-F-, m-F, p-F, α-F, pentafluoro-, p-methyl-, and p-trifluoromethyl-substituted trans-cinnamoyl- α-chymotrypsins has been studied from pH 4 to 8. The deacylation rate constants were found to depend upon the ionization state of a group on the enzyme with an apparent pK in the range 6.3–7.3. The hydrolysis rates of the correspondingly substituted p-nitrophenylcinnamate esters were determined at pH 10.6. Correlation of the data for these model reactions with the corresponding rates of enzyme deacylation suggests that the p-methyl-substituted acylenzyme is about an order of magnitude more reactive than expected while p-trifluoromethyl substitution results in deacylation at a rate 10 times slower than expected. The remaining substituents exert about the anticipated rate effect on deacylation.

scholarly journals Purification and regulatory properties of isocitrate lyase from Escherichia coli ML308

1988 ◽  
Vol 250 (1) ◽  
pp. 25-31 ◽  
Author(s):  
C MacKintosh ◽  
H G Nimmo
Keyword(s):  
Escherichia Coli ◽  
Isocitrate Lyase ◽  
Random Order ◽  
Kinetic Mechanism ◽  
Competitive Inhibitor ◽  
Intact Cells ◽  
Competing Anions ◽  
Effects Of Ph ◽  
Order Of Magnitude

Isocitrate lyase was purified to homogeneity from Escherichia coli ML308. Its subunit Mr and native Mr were 44,670 +/- 460 and 17,000-180,000 respectively. The kinetic mechanism of the enzyme was investigated by using product and dead-end inhibitors of the cleavage and condensation reactions. The data indicated a random-order equilibrium mechanism, with formation of a ternary enzyme-isocitrate-succinate complex. In an attempt to predict the properties of isocitrate lyase in intact cells, the effects of pH, inorganic anions and potential regulatory metabolites on the enzyme were studied. The Km of the enzyme for isocitrate was 63 microM at physiological pH and in the absence of competing anions. Chloride, phosphate and sulphate ions inhibited competitively with respect to isocitrate. Phosphoenolpyruvate inhibited non-competitively with respect to isocitrate, but the Ki value suggested that this effect was unlikely to be significant in intact cells. 3-Phosphoglycerate was a competitive inhibitor. At the concentration reported to occur in intact cells, this metabolite would have a significant effect on the activity of isocitrate lyase. The available data suggest that the Km of isocitrate lyase for isocitrate is similar to the concentration of isocitrate in E. coli cells growing on acetate, about one order of magnitude higher than the Km determined in vitro in the absence of competing anions.

scholarly journals The calculation of transcript flux ratios reveals single regulatory mechanisms capable of activation and repression

2018 ◽  
Vol 115 (50) ◽  
pp. E11604-E11613 ◽  
Author(s):  
Eric A. Galburt
Keyword(s):  
Rate Constants ◽  
Flux Ratio ◽  
Stringent Response ◽  
Kinetic Mechanism ◽  
Regulation Of Transcription ◽  
Sequence Motifs ◽  
Response Factors ◽  
Initiation Rate ◽  
Dna Sequence Motifs ◽  
Kinetics Of

The regulation of transcription allows cells to adjust the rate of RNA polymerases (RNAPs) initiated in a promoter-specific manner. Classically, transcription factors are directed to a subset of promoters via the recognition of DNA sequence motifs. However, a unique class of regulators is recruited directly through interactions with RNAP. Surprisingly, these factors may still possess promoter specificity, and it has been postulated that the same kinetic mechanism leads to different regulatory outcomes depending on a promoter’s basal rate constants. However, mechanistic studies of regulation typically report factor activity in terms of changes in the thermodynamics or kinetics of individual steps or states while qualitatively linking these observations to measured changes in transcript production. Here, I present online calculators that allow for the direct testing of mechanistic hypotheses by calculating the steady-state transcript flux in the presence and absence of a factor as a function of initiation rate constants. By evaluating how the flux ratio of a single kinetic mechanism varies across promoter space, quantitative insights into the potential of a mechanism to generate promoter-specific regulatory outcomes are obtained. Using these calculations, I predict that the mycobacterial transcription factor CarD is capable of repression in addition to its known role as an activator of ribosomal genes. In addition, a modification of the mechanism of the stringent response factors DksA/guanosine 5′-diphosphate 3′-diphosphate (ppGpp) is proposed based on their ability to differentially regulate transcription across promoter space. Overall, I conclude that a multifaceted kinetic mechanism is a requirement for differential regulation by this class of factors.

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.

Modelling the adsorption of phospholipid vesicles to a silicon dioxide surface using Langmuir kinetics

2022 ◽  
Author(s):  
Iad Alhallak ◽  
Peter J. N. Kett
Keyword(s):  
Silicon Dioxide ◽  
Equilibrium Constant ◽  
Rate Constants ◽  
Lipid Vesicles ◽  
Phospholipid Vesicles ◽  
Adsorption And Desorption ◽  
Langmuir Kinetics

The rate constants and equilibrium constant for the adsorption and desorption of lipid vesicles from a SiO2 surface have been determined.

scholarly journals The existence of multiple tetrameric conformers of chicken liver pyruvate carboxylase and their roles in dilution inactivation

1993 ◽  
Vol 290 (2) ◽  
pp. 583-590 ◽  
Author(s):  
P V Attwood ◽  
W Johannssen ◽  
A Chapman-Smith ◽  
J C Wallace
Keyword(s):  
Rate Constants ◽  
Pyruvate Carboxylase ◽  
Electron Microscopic Observation ◽  
Enzyme Concentration ◽  
Enzymic Activity ◽  
Chicken Liver ◽  
Electron Microscopic ◽  
Acetyl Coa ◽  
Degree Of Dissociation ◽  
Tetrameric Structure

The time-dependent loss of enzymic activity and tetrameric structure of chicken liver pyruvate carboxylase (EC 6.4.1.1) after dilution below 2 units/ml was apparently monophasic and first-order. When examined over a range of initial enzyme concentrations, both activity and tetrameric structure decayed to equilibrium levels which were dependent on the initial concentration. The observed rate constants for the loss of enzymic activity (i) showed no apparent dependence on the initial enzyme concentration, and (ii) were of similar magnitude to the corresponding rate constants of dissociation. Computer simulations of the most likely kinetic model suggest that the predominant form of the dissociated enzyme is the monomer. Dilution of pyruvate carboxylase in the presence of the allosteric activator acetyl-CoA largely prevented the subsequent dissociation of the tetrameric molecule. In addition, acetyl-CoA was able to cause a degree of activation and reassociation when added after dilution inactivation had been allowed to occur. Electron-microscopic observation showed the treatment with avidin before dilution markedly decreased the degree of dissociation of the enzyme tetramer. This structure-stabilizing effect of avidin was dependent on preincubation of the concentrated enzyme solution with acetyl-CoA. We propose that, over a range of protein concentrations, the tetrameric enzyme exists in two forms that are in equilibrium, and that acetyl-CoA alters the equilibrium to favour the more compact form.

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