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Enzyme Kinetics01:19

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One example of how cells use the energy contained in electrochemical gradients is demonstrated by glucose transport into cells. The ion vital to this process is sodium (Na+), which is typically present in higher concentrations extracellularly than in the cytosol. Such a concentration difference is due, in part, to the action of an enzyme "pump" embedded in the cellular membrane that actively expels Na+ from a cell. Importantly, as this pump contributes to the high concentration of...
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The theory of catalytically perfect enzymes was first proposed by W.J. Albery and J. R. Knowles in 1976. These enzymes catalyze biochemical reactions at high-speed. Their catalytic efficiency values range from 108-109 M-1s-1. These enzymes are also called 'diffusion-controlled' as the only rate-limiting step in the catalysis is that of the substrate diffusion into the active site. Examples include triose phosphate isomerase, fumarase, and superoxide dismutase.
 
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Determination of Michaelis Constant and Maximum Elimination Rate01:20

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The Michaelis constant (KM) and the theoretical maximum process rate (Vmax) are vital parameters in the Michaelis-Menten equation, central to many biochemical reactions. They provide essential insights into enzyme kinetics and drug metabolism.
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Correlation between Apparent Substrate Affinity and OCT2 Transport Turnover.

Alyscia Cory Severance1, Philip J Sandoval1, Stephen H Wright2

  • 1Department of Physiology, College of Medicine, University of Arizona, Tucson, Arizona.

The Journal of Pharmacology and Experimental Therapeutics
|June 16, 2017
PubMed
Summary
This summary is machine-generated.

Organic cation transporter 2 (OCT2) plays a key role in drug clearance. This study reveals a correlation between substrate affinity and transport rate, suggesting substrate dissociation limits OCT2 transport speed.

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Area of Science:

  • Pharmacology
  • Renal Physiology
  • Drug Transport

Background:

  • Organic cation transporter 2 (OCT2) is crucial for renal drug secretion, mediating the initial uptake of cationic drugs into proximal tubule cells.
  • Accurate kinetic parameters (Jmax/Kt) of OCT2 transport are essential for physiologically-based pharmacokinetic (PBPK) modeling and in vitro-in vivo extrapolation (IVIVE) of drug clearance.
  • The relationship between substrate affinity and maximal transport rate (Jmax) for OCT2 has been underexplored despite the transporter's multispecificity.

Purpose of the Study:

  • To investigate the relationship between the affinity (Kapp) and maximal transport rate (Jmax) of structurally diverse substrates for the OCT2 transporter.
  • To determine if substrate-drug interactions with OCT2 follow a predictable kinetic pattern that can inform drug clearance estimations.
  • To test the hypothesis that substrate dissociation from OCT2 is the rate-limiting step for maximal transport.

Main Methods:

  • Quantification of Jmax and apparent Michaelis constant (Kapp) values for six distinct OCT2 substrates using OCT2-expressing cells.
  • Assessment of trans-stimulation of 1-methyl-4-phenylpyridinium (MPP) uptake following preloading with either high- or low-affinity substrates.
  • Analysis of kinetic data to identify correlations between substrate affinity and maximal transport rates.

Main Results:

  • A strong positive correlation was observed between Jmax and Kapp for OCT2 substrates; high-affinity substrates (low Kapp) exhibited lower Jmax values.
  • Low-affinity substrates (high Kapp) demonstrated significantly higher Jmax values compared to high-affinity substrates.
  • Trans-stimulation experiments confirmed that preloading with low-affinity substrates yielded higher MPP uptake rates than preloading with high-affinity substrates.

Conclusions:

  • The findings support the hypothesis that the dissociation rate of substrates from OCT2 is the rate-limiting step for maximal transport.
  • A systematic relationship exists between OCT2 substrate affinity and transport rate, which can be leveraged for estimating intrinsic clearance (Clint) for drugs lacking transport data.
  • This kinetic understanding of OCT2 is vital for improving predictions of drug pharmacokinetics and optimizing drug development.