scholarly journals The effect of methanol and dioxan on the rates of the β-galactosidase-catalysed hydrolyses of some β-d-galactopyranosides: rate-limiting degalactosylation. The pH-dependence of galactosylation and degalactosylation

1973 ◽  
Vol 133 (1) ◽  
pp. 81-87 ◽  
Author(s):  
Michael L. Sinnott ◽  
Odile M. Viratelle
Keyword(s):  
Steady State ◽  
Ph Dependence ◽  
Methanol Concentration ◽  
The Other ◽  
Fractional Rate ◽  
Rate Of Increase ◽  
Rate Limiting ◽  
Hydrolysis Of

1. The effect of methanol on the β-galactosidase-catalysed hydrolysis of some nitrophenyl β-d-galactopyranosides has been studied under steady-state conditions. 2. The initial fractional rate of increase of kcat. as a function of methanol concentration with 2,4- and 3,5-dinitrophenyl β-d-galactopyranosides, but not with the other substrates studied, indicated that degalactosylation of the enzyme was rate-limiting. 3. The decrease in kcat. at high methanol concentrations for these substrates is considered to arise from causes other than galactosylation becoming rate-limiting. 4. Both galactosylation and degalactosylation of the enzyme require protonation of a group of pKa approx. 9.

scholarly journals Catalysis by acetylcholinesterase in two-hydronic-reactive states. Integrity of deuterium oxide effects and hydron inventories

1992 ◽  
Vol 285 (2) ◽  
pp. 451-460 ◽  
Author(s):  
E Salih
Keyword(s):  
Kinetic Parameters ◽  
Conformational Change ◽  
Rate Constants ◽  
Literature Abstract ◽  
Deuterium Oxide ◽  
Ph Dependence ◽  
Reversible Inhibitor ◽  
Induced Fit ◽  
Rate Limiting ◽  
Hydrolysis Of

Low 2H2O effects (1.0-1.5) for the parameter k(cat.)/Km in the hydrolysis of various substrates by acetylcholinesterase (AcChE) is due to normal 2H2O effects (1.8-2.8) for the parameter k(cat.) and 2H2O effects of 1.0-2.5 for the parameter Km. The analyses and interpretations of 2H2O effects in the literature utilizing the parameter k(cat.)/Km, which led to the proposal of ‘isotope insensitivity’ of the catalytic steps and the hypothesis of a rate-limiting substrate-induced-fit conformational change, are incorrect. Since k(cat.) is the only parameter that can represent the hydron-transfer step solely, the 2H2O effect can most appropriately be evaluated by using this parameter. Calculations and comparison of acylation (k+2) and deacylation (k+3) rate constants show that acylation is rate-determining for most substrates and the improved binding -0.84 to -2.09 kJ/mol (-0.2 to -0.5 kcal/mol) in 2H2O obscures the normal 2H2O effect on k(cat.) when the ratio k(cat.)/Km is utilized. Consistent with this, measurements of the inhibition constant (KI(com.)) for a reversible inhibitor, phenyltrimethylammonium, lead to KI(com.)H2O = 39 +/- 3 microM and KI(com.)2H2O = 24.5 +/- 3.5 microM, an 2H2O effect of 1.59 +/- 0.26. pH-dependence of k(cat.) in 2H2O is subject to variability of the pK(app.) values, as evaluated in terms of the two-hydronic-reactive states (EH and EH2) of AcChE, and is due to an uneven decrease in 2H2O of the kinetic parameters k'cat. for the EH2 state relative to k(cat.) for the EH state, thus leading to variable shifts in pK(app.) values of between 0.5 and 1.2 pH units for this parameter. The observed pH-independent limiting rate constants for k(cat.)/Km(app.) are made to vary between 0.5 and 1.0 in 2H2O by effects on kinetic parameters for the EH2 state, k'cat./K'm varying between 0.2 and 0.7 relative to the EH state, with k(cat.)/Km varying between 0.4 and 1.0. The effects observed on k(cat.)/Km(app.) are ultimately the result of variable effects of 2H2O on k'cat. and K'm for the EH2 state relative to k(cat.) and Km for the EH state of AcChE. These effects are responsible for the variable shifts and more than 0.5 pH unit of the pK(app.) values in 2H2O for pH-k(cat.)/Km profiles. The upward-bowing hydron inventories for k(cat.)/Km are the result of linear hydron inventories for k(cat.) and downward-bowing on Km and are not due to the rate-limiting substrate-induced fit process as claimed in the literature.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Cation-Anion Balance during Potassium and Sodium Absorption by Barley Roots

1963 ◽  
Vol 46 (3) ◽  
pp. 369-386 ◽  
Author(s):  
P. C. Jackson ◽  
H. R. Adams
Keyword(s):  
Steady State ◽  
External Solution ◽  
Potassium Ion ◽  
Sodium Ion ◽  
The Other ◽  
Sodium Absorption ◽  
Specific Rate ◽  
Sodium Ions ◽  
Ion Absorption ◽  
Rate Limiting

Steady-state rates of potassium ion and sodium ion absorption by excised barley roots accompanied by various anions were compared with the rates of anion absorption and the concomitant H+ and base release by the roots. The cation absorption rates were found to be independent of the identities, concentrations, and rates of absorption of the anions of the external solution, including bicarbonate. Absorption of the anion of the salt plus bicarbonate could not account for the cation absorption. H+ is released during cation absorption and base during anion absorption. The magnitude by which one or the other predominates depends on the relative rates of anion and cation absorption under various conditions of pH, cation and anion concentration, and inhibitor concentrations. The conclusion is that potassium and sodium ions are absorbed independently of the anions of the absorption solution in exchange for H+, while anions are exchanged for a base. The H+ release reflects a specificity between K+ and Na+ absorption such that it appears to be H+ exchanged in the specific rate-limiting reactions of the cation absorption.

scholarly journals Variation in the pH-dependent pre-steady-state and steady-state kinetic characteristics of cysteine-proteinase mechanism: evidence for electrostatic modulation of catalytic-site function by the neighbouring carboxylate anion

2003 ◽  
Vol 372 (3) ◽  
pp. 735-746 ◽  
Author(s):  
Syeed HUSSAIN ◽  
Surapong PINITGLANG ◽  
Tamara S. F. BAILEY ◽  
James D. REID ◽  
Michael A. NOBLE ◽  
...  
Keyword(s):  
Steady State ◽  
Cysteine Proteinase ◽  
Ph Dependence ◽  
Catalytic Site ◽  
Rate Determining Step ◽  
Steady State Kinetic ◽  
Pka Value ◽  
Site Function ◽  
Hydrolysis Of ◽  
State Kinetic

The acylation and deacylation stages of the hydrolysis of N-acetyl-Phe-Gly methyl thionoester catalysed by papain and actinidin were investigated by stopped-flow spectral analysis. Differences in the forms of pH-dependence of the steady-state and pre-steady-state kinetic parameters support the hypothesis that, whereas for papain, in accord with the traditional view, the rate-determining step is the base-catalysed reaction of the acyl-enzyme intermediate with water, for actinidin it is a post-acylation conformational change required to permit release of the alcohol product and its replacement in the catalytic site by the key water molecule. Possible assignments of the kinetically influential pKa values, guided by the results of modelling, including electrostatic-potential calculations, and of the mechanistic roles of the ionizing groups, are discussed. It is concluded that Asp161 is the source of a key electrostatic modulator (pKa 5.0±0.1) in actinidin, analogous to Asp158 in papain, whose influence is not detected kinetically; it is always in the ‘on’ state because of its low pKa value (2.8±0.06).

scholarly journals Control of electron transfer in the cytochrome system of mitochondria by pH, transmembrane pH gradient and electrical potential. The cytochromes b-c segment

1981 ◽  
Vol 194 (2) ◽  
pp. 395-406 ◽  
Author(s):  
S Papa ◽  
M Lorusso ◽  
G Izzo ◽  
F Capuano
Keyword(s):  
Steady State ◽  
Electron Flow ◽  
Ph Dependence ◽  
Electrical Potential ◽  
The Other ◽  
Ph Gradient ◽  
Preferential Oxidation ◽  
Submitochondrial Particles ◽  
Cytochrome B562 ◽  
Other Hand

1. A study is presented of the effects of pH, transmembrane pH gradient and electrical potential on oxidoreductions of b and c cytochromes in ox heart mitochondria and ‘inside-out’ submitochondrial particles. 2. Kinetic analysis shows that, in mitochondria at neutral pH, there is a restraint on the aerobic oxidation of cytochrome b566 with respect to cytochrome b562. Valinomycin plus K+ accelerates cytochrome b566 oxidation and retards net oxidation of cytochrome b562. At alkaline pH the rate of cytochrome b566 oxidation approaches that of cytochrome b562 and the effects of valinomycin on b cytochromes are impaired. 3. At slightly acidic pH, oxygenation of antimycin-supplemented mitochondria causes rapid reduction of cytochrome b566 and small delayed reduction of cytochrome b562. Valinomycin or a pH increase in the medium promote reduction of cytochrome b562 and decrease net reduction of cytochrome b566. 4. Addition of valinomycin to mitochondria and submitochondrial particles in the respiring steady state causes, at pH values around neutrality, preferential oxidation of cytochrome b566 with respect to cytochrome b562. The differential effect of valinomycin on oxidation of cytochromes b566 and b562 is enhanced by substitution of 1H2O of the medium with 2H2O and tends to disappear as the pH of the medium is raised to alkaline values. 5. Nigericin addition in the aerobic steady state causes, both in mitochondria and submitochondrial particles, preferential oxidation of cytochrome b562 with respect to cytochrome b566. This is accompanied by c cytochrome oxidation in mitochondria but c cytochrome reduction in submitochondrial particles. 6. In mitochondria as well as in submitochondrial particles, the aerobic transmembrane potential (delta psi) does not change by raising the pH of the external medium from neutrality to alkalinity. The transmembrane pH gradient (delta pH) on the other hand, decrease slightly. 7. The results presented provide evidence that the delta psi component of the aerobic delta microH+ (the sum of the proton chemical and electrical activities) exerts a pH-dependent constraint on forward electron flow from cytochrome b566 to cytochrome b562. This effect is explained as a consequence of anisotropic location of cytochromes b566 and b562 in the membrane and the pH-dependence of the redox function of these cytochromes. Transmembrane delta pH, on the other hand, exerts control on electron flow from cytochrome b562 to c cytochromes.

scholarly journals Using pH dependence for understanding mechanisms in electrochemical CO reduction

2021 ◽  
Author(s):  
Georg Kastlunger ◽  
Lei Wang ◽  
Nitish Govindarajan ◽  
Hendrik H. Heenen ◽  
Stefan Ringe ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Ph Dependence ◽  
The Other ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Other Hand ◽  
Alkaline Conditions ◽  
Co Reduction ◽  
Rate Limiting ◽  
Electrolyte Ph

Utilizing electrochemical conversion of CO(2) into hydrocarbons and oxygenates is envisioned as a promising path towards closing the carbon cycle in modern technology. To this day, however, the exact reaction mechanisms towards the plethora of single and multi-carbon products on Cu electrodes are still disputed. This uncertainty even extends to the rate-limiting step of the respective reactions. Since multi-carbon products do not show a dependence on the electrolyte pH in neutral and alkaline media, CO dimerization on the Cu surface has been proposed as the rate-limiting step. However, other elementary steps would lead to the same pH dependence, namely the proton-electron transfer to *CO followed by subsequent coupling or the protonation of the *OCCO dimer. The pH dependence of methane production on the other hand suggests that the rate limiting step is located beyond the first proton-electron transfer to *CO. In order to conclusively identify the rate limiting steps in CO reduction, we analyzed the mechanisms on the basis of constant potential DFT calculations, CO reduction experiments on Cu at varying pH values (3 - 13) and fundamental rate theory. We find that, even in acidic media, the reaction rate towards multi-carbon products is nearly unchanged on an SHE potential scale, which indicates that its rate limiting step does not involve a proton donor. Hence, we deduce that the rate limiting step can indeed only consist of the coupling of two CO molecules on the surface, both in acidic and alkaline conditions. For methane, on the other hand, the rate-limiting step changes with the electrolyte pH from the first protonation step in acidic/neutral conditions to a later step in alkaline conditions. Finally, based on an in-depth kinetic analysis, we conclude that the pathway towards CH4 involving a surface combination of *CO and *H is unlikely, since it is unable to reproduce the measured current densities and Tafel slopes.

Antimonite regulation of the ATPase activity of ArsA, the catalytic subunit of the arsenical pump

2001 ◽  
Vol 360 (3) ◽  
pp. 589-597 ◽  
Author(s):  
Adrian R. WALMSLEY ◽  
Tongqing ZHOU ◽  
M. Ines BORGES-WALMSLEY ◽  
Barry P. ROSEN
Keyword(s):  
Steady State ◽  
Complex Formation ◽  
Atpase Activity ◽  
Ternary Complex ◽  
Catalytic Subunit ◽  
Product Release ◽  
Ternary Complex Formation ◽  
Atpase Reaction ◽  
Rate Limiting ◽  
Hydrolysis Of

The ArsA ATPase is the catalytic subunit of the pump protein, coupling the hydrolysis of ATP to the movement of arsenicals and antimonials through the membrane-spanning ArsB protein. Previously, we have shown the binding and hydrolysis of MgATP to ArsA to be a multi-step process in which the rate-limiting step is an isomerization between different conformational forms of ArsA. This isomerization occurs after product release, at the end of the ATPase reaction, and involves the return of the ArsA to its original conformation, which can then bind MgATP. ArsA possesses an allosteric site for antimonite [Sb(III)], the binding of which elevates the steady-state ATPase activity. We have used a transient kinetics approach to investigate the kinetics of ternary complex formation that lead to an enhancement in the ATPase activity. These studies revealed that ArsA exists in at least two conformational forms that differ in their ligand binding affinities, and that ATP favours one form and Sb(III) the other. Ternary complex formation is rate-limited by a slow transition between these conformational forms, leading to a lag in attaining maximal steady-state activity. Sb(III) enhances the steady-state ATPase activity by inducing rapid product release, allowing ArsA to adopt a conformation that can bind MgATP for the next catalytic cycle. In the presence of Sb(III), ArsA avoids the rate-limiting isomerization at the end of the ATPase reaction and ATP hydrolysis becomes rate-limiting for the reaction. The binding of Sb(III) probably results in more effective pumping of the substrates from the cell by enhancing the rate of efflux.

Transient-phase studies of a trypsin-catalyzed reaction

1970 ◽  
Vol 48 (12) ◽  
pp. 1793-1802 ◽  
Author(s):  
H. P. Kasserra ◽  
K. J. Laidler
Keyword(s):  
Steady State ◽  
Ph Dependence ◽  
Enzyme Concentration ◽  
Alcohol Concentration ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Steady State Kinetics ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Nitrophenyl Ester

The stopped-flow technique has been used to study the pre-steady-state kinetics of the hydrolysis of N-carbobenzoxy-L-alanine-p-nitrophenyl ester catalyzed by trypsin. By working under conditions such that the enzyme concentration is much greater than that of the substrate, it has been possible to measure [Formula: see text] the rate constant for the conversion of the enzyme-substrate complex into the acyl enzyme. The pH dependence of [Formula: see text] reveals a pKb′ value of 6.9 for the conversion of complex into acyl enzyme, in agreement with deductions from steady-state investigations. The pH dependence of [Formula: see text] (equal to k−1 + k2)/k1) has also been determined. The results provide direct evidence for the existence of an enzyme-substrate complex for this reaction.The work has been done in various mixtures of water and isopropyl alcohol. The logarithms of the rate constants [Formula: see text] and [Formula: see text] vary linearly with 1/D, showing a decrease with increasing alcohol concentration; [Formula: see text] increases with alcohol concentration. The solvent results suggest that addition of alcohol affects the hydrophobic bonding in the protein and leads to unfolding of the enzyme.

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