scholarly journals Resistance to inactivation by EGTA of the enzyme-substrate and enzyme-phosphate complexes of alkaline phosphatase

1987 ◽  
Vol 244 (3) ◽  
pp. 781-785 ◽  
Author(s):  
S J Pike ◽  
R G Duggleby
Keyword(s):  
Alkaline Phosphatase ◽  
Steady State ◽  
Chelating Agent ◽  
Enzyme Substrate ◽  
Steady State Kinetic ◽  
Nitrophenyl Phosphate ◽  
Comparison Of The Results ◽  
State Kinetic ◽  
Substrate Concentrations ◽  
Stability Measurements

Bovine intestinal mucosal alkaline phosphatase is inactivated by the chelating agent EGTA. Several concentrations of the enzyme were incubated with EGTA and a range of concentrations of the substrate p-nitrophenyl phosphate to determine the substrate concentration as a function of time. As predicted by a recently developed theory [Duggleby (1986) J. Theor. Biol. 123, 67-80], catalysis ceases before all substrate is exhausted. An analysis of these final substrate concentrations according to the theory revealed that, whereas the free enzyme is unstable, the effect of EGTA is counteracted when either the substrate or product (phosphate) is bound. Comparison of the results with those obtained by direct stability measurements and steady-state kinetic experiments gave a qualitatively and quantitatively consistent body of evidence in support of this interpretation.

scholarly journals Interpretation of the Kinetics of Consecutive Enzyme-catalysed Reactions: Studies on the Arginase-Ornithine Carbamoyltransferase System

1982 ◽  
Vol 35 (2) ◽  
pp. 137 ◽  
Author(s):  
RD Teasdale ◽  
PD Jeffrey ◽  
PW Kuchel ◽  
LW Nichol
Keyword(s):  
Steady State ◽  
Ornithine Carbamoyltransferase ◽  
Enzyme Substrate ◽  
Steady State Kinetic ◽  
Kinetic Mechanisms ◽  
Heterogeneous Association ◽  
Kinetics Of ◽  
State Kinetic ◽  
Enzymic Assay

A method that permits the use of measurements on the concentration of the intermediate in a coupled enzymic assay in determining the presence or absence of an interaction between the enzymes is presented. The method is shown to be closely analogous to a previously formulated procedure involving the determination of the rate of production of the final product of such a sequence and is shown to be applicable regardless of the complexity of the operative kinetic mechanisms, provided it may be assumed that all enzyme-substrate complexes are in the steady-state. Kinetic results obtained with the arginase--ornithine carbamoyltransferase couple, in which the intermediate ornithine is monitored, are examined in these terms to conclude that no heterogeneous association is operative between the enzymes.

scholarly journals The Complete Pathway for ERK2-catalyzed Reaction

2007 ◽  
Vol 282 (38) ◽  
pp. 27678-27684 ◽  
Author(s):  
Zhi-Xin Wang ◽  
Jia-Wei Wu
Keyword(s):  
Steady State ◽  
Detailed Balance ◽  
Kinetic Studies ◽  
Kinetic Mechanism ◽  
Initial Reaction ◽  
Enzymatic Mechanism ◽  
Enzyme Substrate ◽  
Steady State Kinetic ◽  
Mitogen Activated Protein ◽  
State Kinetic

In the present study, the enzymatic mechanism of ERK2 is re-examined by a combination of steady-state kinetic studies in the absence and presence of viscosogenic agents. Kinetic studies carried out in various concentrations of sucrose revealed that both kcat and kcat/Km for either ATP or EtsΔ138 were highlysensitive to solvent viscosity, suggesting that the rapid equilibrium assumption is not valid for the phosphorylation of protein substrate by ERK2. Furthermore, the kinetic analysis with the minimal random Bi Bi reaction mechanism is shown to be inconsistent with the principle of the detailed balance. This inconsistent calculation strongly suggests that there is isomerization of the enzyme-substrate ternary complex. The viscosity-dependent steady-state kinetic data are combined to establish a kinetic mechanism for the ERK2-catalyzed reaction that predicts initial reaction velocities under varying concentrations of ATP and substrate. These results complement previous structure-function studies of mitogen-activated protein kinases and provide important insight for mechanistic interpretation of the kinase functions.

scholarly journals Phenothiazines are slowly oxidizable substrates of horseradish peroxidase

2011 ◽  
Vol 57 (5) ◽  
pp. 544-553 ◽  
Author(s):  
T.V. Rogozhina ◽  
V.V. Rogozhin
Keyword(s):  
Steady State ◽  
Horseradish Peroxidase ◽  
Reaction Medium ◽  
Substrate Oxidation ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Steady State Kinetic ◽  
State Kinetic ◽  
Full Conversion ◽  
Peroxidase Oxidation

Reactions of peroxidase oxidation of triftazine and thioproperazine have been investigated in the presence of horseradish peroxidase using steady state kinetic methods. It has been shown that phenothiazines are slowly oxidizable substrates for horseradish peroxidase. kcat and Km values have been determined in the range of pH from 4.5 to 7.5. The study of co-oxidation of phenothiazines and o-dianisidine (ODN) revealed that in the presence of aminazine and ODN in the reaction medium both substances follow sequential oxidation. ODN oxidation was not observed until full conversion of aminazine. At pH 4.5-5.5 thioproperazine bound to the enzyme-substrate complex and caused a nticompetitive inhibition of peroxidase. At pH>5.5 sequential substrate oxidation with preferential thioproperazine conversion occurred. In the range of pH from 4.5 to 7.5 triftazine did not influence ODN oxidation.

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