scholarly journals The Experimentally Determined Velocity of Catalysis could be Higher in the Absence of Sequestration

2019 ◽  
pp. 1-12
Author(s):  
Ikechukwu I. Udema ◽  
Abraham Olalere Onigbinde
Keyword(s):  
Maximum Velocity ◽  
Order Rate Constant ◽  
Free Enzyme ◽  
Theoretical Research ◽  
Coefficient Of Determination ◽  
First Order ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Pseudo First Order ◽  
The Relationship

Background: It is not unusual to observe calculated “total” free enzyme ([E]) in enzyme catalysed reaction, but this should include total enzyme-substrate complex ([EST]) which accounts for sequestration. Objectives: 1) To show indirectly that the velocities of catalytic action can be higher than experimentally observed velocities without sequestration and 2) redefine the relationship between velocity of hydrolysis with Michaelian enzyme and [E], where concentration of substrate, [ST] <  Michaelis-Menten constant, KM. Methods: A theoretical research and experimentation using Bernfeld method to determine velocities of amylolysis with which to mathematically calculate [EST] and the enzyme-substrate complex ([ES]) prepared for product, P, formation. Results: The [EST] is < [E]; [EST] and pseudo-first order constant, k decreased with increasing [ST] and increased with increasing concentration of enzyme [ET] while velocity amylolysis, v and maximum velocity of amylolysis, vmax expectedly increased with increasing [ET] and [ST]. Conclusion: The fact is that the [EST] is lower than what is usually referred to as free enzyme ([ET] - [ES]). Therefore, if the additional part of [EST] dissociated into product within the duration of assay, the velocity of amylolysis could be higher. The most important outcome and corollary when [KM] > [ST] is that v a 1/[E], v a [E][ST] and a quadratic relationship exists between pseudo-first order rate constant and maximum velocity of amylolysis; separately, v is not a [E] and if v a [ST] (if v/[ST] is constant with coefficient of determination = 1), then KM is not applicable.

Total Enzyme-substrate Complex Which Includes Product-destined Enzyme-substrate Complex

2019 ◽  
pp. 1-7
Author(s):  
Ikechukwu I. Udema
Keyword(s):  
Complex Formation ◽  
Maximum Velocity ◽  
Free Enzyme ◽  
Theoretical Research ◽  
Substrate Complex ◽  
Continuous Formation ◽  
Enzyme Substrate ◽  
The Difference ◽  
Total Enzyme

Background: There is no much interest in the determination of total enzyme-substrate complex concentration ([ES]T) which includes undissociated ES that is unaccounted for unlike the usual ES destined for transformation into free enzyme and product or substrate. The reason is speculatively as a result of the lack of awareness of such possibility via sequestration. Objectives: 1) To derive on the basis of both reverse – and standard – quasi-steady – state assumptions equations for the determination of [ES]T which is not restricted to the complex which dissociates to product/substrate and free enzyme and 2) quantitate the value of [ES]T. Methods: A theoretical research and experimentation using Bernfeld method to determine velocities of amylolysis with which to calculate relevant parameters. Results: The [EST] is < [E] ( i. e. [ET] - [ES]); [EST] decreased with increasing [ST] and increased with increasing concentration of enzyme [ET] while the velocity of amylolysis, v and maximum velocity of amylolysis, vmax expectedly increased with increasing [ET] and [ST]. Conclusion: The equations for the determination of the total enzyme-substrate complex, free enzyme without any complex formation before and after dissociation of enzyme-complex into product and/or substrate and free enzyme were derived. The difference, [ET] - [ES] is a heterogeneous mixture of undissociated ES and free enzyme without any complex formation. This is the case because [ES] which dissociates into product is only a part of the total enzyme-substrate complex. There is a continuous formation of ES during and at the expiry of the duration of assay as long as there is no total substrate depletion.

scholarly journals Photoenzymatic Repair of Ultraviolet Damage in DNA

1962 ◽  
Vol 45 (4) ◽  
pp. 725-741 ◽  
Author(s):  
Claud S. Rupert
Keyword(s):  
Heavy Metals ◽  
Quantum Yield ◽  
Kinetic Studies ◽  
Order Rate Constant ◽  
Reaction Scheme ◽  
First Order ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Exponential Decrease ◽  
Ultraviolet Damage

The photoenzyme from bakers' yeast which repairs ultraviolet-inactivated transforming DNA is mechanically bound to ultraviolet-irradiated DNA in the dark, but not to unirradiated DNA. In the bound condition it is stabilized against inactivation by heat and heavy metals. Both the mechanical binding and stabilization are eliminated by illumination. These observations are consistent with the reaction scheme suggested by kinetic studies, in which the enzyme combines with the ultraviolet lesions in DNA and the complex absorbs light, producing repair and subsequent liberation of the enzyme. The approximately exponential decrease of heat stabilization during illumination gives the first order rate constant for the light-dependent step at the corresponding light intensity. This quantity in turn sets limits on the possible magnitude of the molar absorption coefficient of the enzyme-substrate complex and on the quantum yield of the process.

scholarly journals Exo-Site Affinity Labeling of Human α(CLOTTING) and β/γ(NONCLOTTING) m[o-2-CHLORO-5-Sulfonylfluorophenylureido) Phenoxybutoxy] Benzamidine (mCP(PBA)-F)

1977 ◽  
Author(s):  
D.H. Bing ◽  
J.W. Fenton ◽  
M. Cory
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Order Rate Constant ◽  
Affinity Labeling ◽  
First Order ◽  
Enzyme Substrate ◽  
Pseudo First Order ◽  
A Chain ◽  
Substrate Kinetics ◽  
Labeling Pattern

Human α-thrombin was selectively and irreversibly inactivated with mCP(PBA)-F (pseudo first order rate constant k1=3.7x10-3 sec-1 23°). The initial velocities of inactivation followed simple enzyme-substrate kinetics (Km (app)=72.7 μM, k2=3x10-3 sec-1). mCP(PBA)-F was determined to be an affinity labeling reagent for both α and β/γ-thrombins (7% α, 20% β and 78% γ) as (1)[3H]mCP(PBA)-F was stochiometrically incorporated (1.07 mole/mole for α thrombin and 1.19 mole/mole for β/γ-thrombin), (2) [14C]iPr2P-F inactivated α and β/γ-thrombin did not react with mCP(PBA)-F, and (3) the analog o-(2-chloro-5-sulfonylf luorophenylureido)phenoxy benzene lacking the benzamidine, only caused 14% inactivation of α-thrombin. [14C]iPr2P-F was only found on the B chain (Mr 32,000) but [3H]m(PBA)-F label was distributed 20% on the A chain and 80% on the B chain in reduced and alkylated α-thrombin electrophoresed in 10% PAGE-SDS-6 M urea. The labeling pattern of β/γ-thrombin with [14C]iPr2-F and [3H]mCP(PBA)-F revealed that 14C label was on the B chain active site fragments of β-thrombin (Mr ≈ 23,000) and γ-thrombin (Mr ≈ 12,000), but the 3H label was on 20% the A chain and 80% on fragments of Mr 6000-10,000. The results suggest that mCP(PBA)-F reacts with residues exclusive of the active site serine within ~ 10 Å radius of the anionic binding site.(Supported by NIH Grants AI 11231, AM 17351, CA 17376, HL 13160 and HL 18825)

scholarly journals Photocatalytic degradation of indigo carmine by ZnO photocatalyst under visible light irradiation

2017 ◽  
Vol 14 (3) ◽  
pp. 582-587
Author(s):  
Baghdad Science Journal
Keyword(s):  
Zinc Oxide ◽  
Photocatalytic Degradation ◽  
Indigo Carmine ◽  
Catalyst Loading ◽  
Order Kinetic ◽  
Kinetic Reaction ◽  
First Order ◽  
Pseudo First Order ◽  
The Relationship ◽  
Influential Parameters

In this work, the photocatalytic degradation of indigo carmine (IC) using zinc oxide suspension was studied. The effect of influential parameters such as initial indigo carmine concentration and catalyst loading were studied with the effect of Vis irradiation in the presence of reused ZnO was also investigated. The increased in initial dye concentration decreased the photodegradation and the increased catalyst loading increased the degradation percentage and the reused-ZnO exhibits lower photocatalytic activity than the ZnO catalyst. It has been found that the photocatalytic degradation of indigo carmine obeyed the pseudo-first-order kinetic reaction in presence of zinc oxide. This was found from plotting the relationship between ln (C0/Ct) and irradiation the rate constant of the process.UV- spectrophotometer was used to study the indigo carmine photodegradation.

scholarly journals Fructose 1,6-bisphosphate aldolase from rabbit muscle. The isomerization of the enzyme-dihydroxyacetone phosphate complex

1977 ◽  
Vol 167 (2) ◽  
pp. 361-366 ◽  
Author(s):  
E Grazi ◽  
M Blanzieri
Keyword(s):  
Competitive Inhibitor ◽  
Dihydroxyacetone Phosphate ◽  
Rabbit Muscle ◽  
Keto Form ◽  
First Order ◽  
Phosphate Complex ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Dead End ◽  
Fructose 1,6 Bisphosphate

The formation and dissociation of the aldolase-dihydroxyacetone phosphate complex were studied by following changes in A240 [Topper, Mehler & Bloom (1957), Science 126, 1287-1289]. It was shown that the enzyme-substrate complex (ES) slowly isomerizes according to the following reaction: (formula: see text) the two first-order rate constants for the isomerization step being k+2 = 1.3s-1 and k-2 = 0.7s-1 at 20 degrees C and pH 7.5. The dissociation of the ES complex was provoked by the addition of the competitive inhibitor hexitol 1,6-bisphosphate. At 20 degrees C and pH 7.5, k+1 was 4.7 X 10(6)M-1-S-1 and k-1 was 30s-1. Both the ES and the ES* complexes react rapidly with 1.7 mM-glyceraldehyde 3-phosphate, the reaction being practically complete in 40 ms. This shows that the ES* complex is not a dead-end complex. Evidence was also provided that aldolase binds and utilizes only the keto form of dihydroxyacetone phosphate.

Kinetics of reconstitution of apotyrosinase by copper

1990 ◽  
Vol 68 (2) ◽  
pp. 476-479
Author(s):  
Donald C. Wigfield ◽  
Douglas M. Goltz
Keyword(s):  
Rate Constant ◽  
Copper Concentration ◽  
Copper Ions ◽  
Copper Ion ◽  
Order Rate Constant ◽  
First Order ◽  
Order Rate ◽  
Pseudo First Order ◽  
Rate Limiting ◽  
Kinetics Of

The kinetics of the reconstitution reaction of apotyrosinase with copper (II) ions are reported. The reaction is pseudo first order with respect to apoenzyme and the values of these pseudo first order rate constants are reported as a function of copper (II) concentration. Two copper ions bind to apoenzyme, and if the second one is rate limiting, the kinetically relevant copper concentration is the copper originally added minus the amount used in binding the first copper ion to enzyme. This modified copper concentration is linearly related to the magnitude of the pseudo first order rate constant, up to a copper concentration of 1.25 × 10−4 M (10-fold excess), giving a second order rate constant of 7.67 × 102 ± 0.93 × 102 M−1∙s−1.Key words: apotyrosinase, copper, tyrosinase.

scholarly journals Acrolein, an irreversible active-site-directed inhibitor of deoxyribose 5-phosphate aldolase?

1976 ◽  
Vol 153 (2) ◽  
pp. 495-497 ◽  
Author(s):  
D C Wilton
Keyword(s):  
Schiff Base ◽  
Rate Constant ◽  
Active Site ◽  
Order Rate Constant ◽  
Lysine Residue ◽  
Prolonged Incubation ◽  
Enzyme Active Site ◽  
First Order ◽  
Substrate Analogue ◽  
Pseudo First Order

The enzyme deoxyribose 5-phosphate aldolase was irreversibly inactivated by the substrate analogue acrolein with a pseudo-first-order rate constant of 0.324 min-1 and a Ki (apparent) of 2.7 × 10(-4) m. No inactivation was observed after prolonged incubation with the epoxide analogues glycidol phosphate and glycidaldehyde. It is suggested that the acrolein is first activated by forming a Schiff base with the enzyme active-site lysine residue and it is the activated inhibitor that reacts with a suitable-active-site nucleophile.

An automated approach to characterize, in simple kinetic terms, the generation and inactivation of thrombin and the interaction of fibrinogen with thrombin.

1988 ◽  
Vol 34 (10) ◽  
pp. 1971-1975 ◽  
Author(s):  
D R Hoak ◽  
S K Banerjee ◽  
G Kaldor
Keyword(s):  
Prothrombin Time ◽  
Rate Constant ◽  
Thrombin Generation ◽  
Order Rate Constant ◽  
Clinical Use ◽  
First Order ◽  
Pathological Conditions ◽  
Pseudo First Order ◽  
Instantaneous Concentration

Abstract Here, we used a fully automated, computer-directed centrifugal analyzer (which permitted simultaneous turbidimetry and calculation of results) and purified thrombin, fibrinogen, and various inhibitors to study clot formation. The Km and Vm for these reactions were useful in detecting and partly characterizing anticoagulants. We also explored the generation and inactivation of thrombin, using the two-stage prothrombin time and antithrombin activity tests. The amount of thrombin instantaneously generated and inactivated was monitored under artificially created pathological conditions. The pseudo-first-order rate constant for thrombin generation and inactivation and the instantaneous concentration of enzymatically active and inactive thrombin were used in the characterization of these conditions. We believe this approach is suitable for routine clinical use.

The influence of pH and inhibitors on the kinetics of enzyme reactions involving two intermediates

1967 ◽  
Vol 45 (5) ◽  
pp. 539-546 ◽  
Author(s):  
Harvey Kaplan ◽  
Keith J. Laidler
Keyword(s):  
Steady State ◽  
Ph Dependence ◽  
Free Enzyme ◽  
State Equations ◽  
Enzyme Reactions ◽  
Substrate Complex ◽  
Rate Determining Step ◽  
Enzyme Substrate ◽  
Special Cases ◽  
Influence Of Ph

General steady-state equations are worked out for enzyme reactions which occur according to the scheme [Formula: see text]Equations showing the pH dependence of the kinetic parameters are developed in a form which distinguishes between essential and nonessential ionizing groups. The pK dependence of [Formula: see text], the second-order constant extrapolated to zero substrate constant, gives pK values for groups which ionize on the free enzyme, but reveals such a pK only if the corresponding group is also involved in the breakdown of the Michaelis complex. General steady-state equations are also developed for the case in which an inhibitor can combine with the free enzyme, the enzyme–substrate complex, and also a second intermediate (e.g. an acyl enzyme). The equations are given in a form that is convenient for analyzing the experimental results, and a number of special cases are considered. It is shown how the type of inhibition depends not only on the nature of the inhibitor but also on that of the substrate, an important factor being the rate-determining step of the reaction. Examples of the various kinds of behavior are given.

Kinetics and mechanisms of oxidations by metal ions. Part IV. Oxidation of phenylphosphinic acid by aquavanadium(V) ions

1985 ◽  
Vol 63 (3) ◽  
pp. 663-666 ◽  
Author(s):  
Raj Narain Mehrotra
Keyword(s):  
Acidic Solution ◽  
Active Form ◽  
Equilibrium Mixture ◽  
Order Rate Constant ◽  
First Order ◽  
Inactive Form ◽  
Pseudo First Order ◽  
Independent Path ◽  
Kinetics Of ◽  
Vanadium Ion

The kinetics of the oxidation of phenylphosphinic acid by quinquevalent vanadium ion have been investigated in aqueous perchlorate media under pseudo-first order conditions (phenylphosphinic acid in excess). The reaction has a first order dependence in [V(V)] and [phenylphosphinic acid] and the observed pseudo-first order rate constant kobs is given by kobs = a + b[H+].The acid-independent path is considered to be due to the reaction between VO2+ (aq.) and C6H5P:(OH)2, the active form of phenylphosphinic acid, while the reaction between V(OH)32+ (aq.) and C6H5P(O)(OH)H, the inactive form of phenylphosphinic acid, is considered to explain the acid-dependent path. Phenylphosphinic acid in aqueous acidic solution is known to exist as an equilibrium mixture of the active and inactive forms. The composite activation and thermodynamic parameters associated with the constants a and b are reported.

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