scholarly journals A linear equation that describes the steady-state kinetics of enzymes and subcellular particles interacting with tightly bound inhibitors

1972 ◽  
Vol 127 (2) ◽  
pp. 321-333 ◽  
Author(s):  
Peter J. F. Henderson
Keyword(s):  
Steady State ◽  
Dose Response ◽  
Significant Proportion ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
High Affinity ◽  
Mechanism Of Inhibition ◽  
Steady State Kinetics ◽  
Linear Plot ◽  
Kinetics Of

When an enzyme exhibits a high affinity for an inhibitor, the steady-state analysis of the mechanism is complicated by the non-linearity of normal dose–response plots or of reciprocal replots. It is shown here that dose–response measurements generate a linear plot of inhibitor concentration divided by degree of inhibition against velocity without inhibitor divided by velocity with inhibitor; the concentration of enzyme may be derived from the extrapolated intercept of such plots, and the mechanism of inhibition from replots of the variation of the slope with substrate concentration. The limiting cases where virtually all inhibitor molecules are bound or virtually all are free are described, together with the situation when a significant proportion of the substrate becomes bound. This type of analysis indicates that the inhibitors of oxidative phosphorylation, rutamycin and bongkrekic acid, are tightly bound to rat liver mitochondria.

scholarly journals Effect of Copper on the Mitochondrial Carnitine/Acylcarnitine Carrier Via Interaction with Cys136 and Cys155. Possible Implications in Pathophysiology

Molecules ◽  
2020 ◽  
Vol 25 (4) ◽  
pp. 820
Author(s):  
Nicola Giangregorio ◽  
Annamaria Tonazzi ◽  
Lara Console ◽  
Mario Prejanò ◽  
Tiziana Marino ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Dose Response ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Response Analysis ◽  
Native Protein ◽  
Mechanism Of Inhibition ◽  
Ic50 Values ◽  
Effect Of Copper ◽  
Substrate Protection

The effect of copper on the mitochondrial carnitine/acylcarnitine carrier (CAC) was studied. Transport function was assayed as [3H]carnitine/carnitine antiport in proteoliposomes reconstituted with the native protein extracted from rat liver mitochondria or with the recombinant CAC over-expressed in E. coli. Cu2+ (as well as Cu+) strongly inhibited the native transporter. The inhibition was reversed by GSH (reduced glutathione) or by DTE (dithioerythritol). Dose-response analysis of the inhibition of the native protein was performed from which an IC50 of 1.6 µM for Cu2+ was derived. The mechanism of inhibition was studied by using the recombinant WT or Cys site-directed mutants of CAC. From the dose-response curve of the effect of Cu2+ on the recombinant protein, an IC50 of 0.28 µM was derived. Inhibition kinetics revealed a non-competitive type of inhibition by Cu2+. However, a substrate protection experiment indicated that the interaction of Cu2+ with the protein occurred in the vicinity of the substrate-binding site. Dose-response analysis on Cys mutants led to much higher IC50 values for the mutants C136S or C155S. The highest value was obtained for the C136/155S double mutant, indicating the involvement of both Cys residues in the interaction with Cu2+. Computational analysis performed on the WT CAC and on Cys mutants showed a pattern of the binding energy mostly overlapping the binding affinity derived from the dose-response analysis. All the data concur with bridging of Cu2+ with the two Cys residues, which blocks the conformational changes required for transport cycle.

scholarly journals Kinetics of suicide substrates. Practical procedures for determining parameters

1985 ◽  
Vol 227 (3) ◽  
pp. 843-849 ◽  
Author(s):  
S G Waley
Keyword(s):  
Steady State ◽  
Kinetic Parameters ◽  
Experimental Error ◽  
Simulated Data ◽  
Steady State Kinetics ◽  
Linear Plot ◽  
Series Of Experiments ◽  
Kinetics Of ◽  
Competing Reactions ◽  
Suicide Substrates

Many clinically important or mechanistically interesting inhibitors react with enzymes by a branched pathway in which inactivation of the enzyme and formation of product are competing reactions. The steady-state kinetics for this pathway [Waley (1980) Biochem. J. 185, 771-773] gave equations for progress curves that were cumbersome. A convenient linear plot is now described. The time (t1/2) for 50% inactivation of the enzyme (this is also the time for 50% formation of product), or for 50% loss of substrate, is measured in a series of experiments in which the concentration of inhibitor, [I]0, is varied; in these experiments the ratio of the concentration of enzyme to the concentration of inhibitor is kept fixed. Then a plot of [I]0 X t1/2 against [I]0 is linear, and the kinetic parameters can be found from the slope and intercept. Furthermore, simplifications of the equations for progress curves are described that are valid when the concentration of inhibitors is high, or is low, or when the extent of reaction is low. The use of simulated data has shown that the recommended methods are not unduly sensitive to experimental error.

scholarly journals Kinetics of Plasmodium falciparum thymidylate synthase: interactions with high-affinity metabolites of 5-fluoroorotate and D1694.

1996 ◽  
Vol 40 (7) ◽  
pp. 1628-1632 ◽  
Author(s):  
M Hekmat-Nejad ◽  
P K Rathod
Keyword(s):  
Plasmodium Falciparum ◽  
Steady State ◽  
Thymidylate Synthase ◽  
Biochemical Parameters ◽  
Potent Inhibitor ◽  
Antimalarial Activity ◽  
High Affinity ◽  
Thymidylate Synthases ◽  
Steady State Kinetics ◽  
Kinetics Of

Consistent with a proposed mechanism for the potent antimalarial activity of 5-fluoroorotate, 5-fluoro-2'-deoxyuridylate inhibited Plasmodium falciparum thymidylate synthase with a Ki of 2 nM. Steady-state kinetics revealed no significant differences between malarial and mammalian thymidylate synthases. Thus, additional biochemical parameters must underlie the selective antimalarial activity of 5-fluoroorotate. A polyglutamylated folate analog, D1694-(glu)4, was also a potent inhibitor of malarial thymidylate synthase (Kis = 1.5 nM).

scholarly journals The Steady State Maintenance of Accumulated Ca++ in Rat Liver Mitochondria

1965 ◽  
Vol 240 (6) ◽  
pp. 2712-2720
Author(s):  
Zdenek Drahota ◽  
Ernesto Carafoli ◽  
Carlo S. Rossi ◽  
Robert L. Gamble ◽  
Albert L. Lehninger
Keyword(s):  
Steady State ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria
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