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Related Concept Videos

Introduction to Enzyme Kinetics01:19

Introduction to Enzyme Kinetics

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Enzyme kinetics studies the rates of biochemical reactions. Scientists monitor the reaction rates for a particular enzymatic reaction at various substrate concentrations. Additional trials with inhibitors or other molecules that affect the reaction rate may also be performed.
The experimenter can then plot the initial reaction rate or velocity (Vo) of a given trial against the substrate concentration ([S]) to obtain a graph of the reaction properties. For many enzymatic reactions involving a...
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Enzyme Kinetics01:19

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Enzymes speed up reactions by lowering the activation energy of the reactants. The speed at which the enzyme turns reactants into products is called the rate of reaction. Several factors impact the rate of reaction, including the number of available reactants. Enzyme kinetics is the study of how an enzyme changes the rate of a reaction.
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Catalytically Perfect Enzymes01:07

Catalytically Perfect Enzymes

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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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Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions
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Characterizing Bi-substrate Enzyme Kinetics at High Resolution by 2D-ITC.

Yun Wang1, Anthony K Mittermaier1

  • 1Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec H3A 0B8, Canada.

Analytical Chemistry
|September 13, 2021
PubMed
Summary

A new 2D-ITC method rapidly characterizes enzyme kinetics, measuring reaction velocity across two substrate concentrations in one experiment. This approach reveals novel allosteric regulation insights, like phenylalanine alleviating ATP inhibition in pyruvate kinase.

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Area of Science:

  • Biochemistry
  • Enzyme kinetics
  • Biophysical chemistry

Background:

  • Isothermal titration calorimetry (ITC) is valuable for real-time reaction enthalpy measurement.
  • Characterizing bi-substrate enzymes using traditional ITC is experimentally demanding, requiring numerous independent experiments.
  • Existing methods are time-consuming and costly for comprehensive kinetic analysis.

Purpose of the Study:

  • To develop a novel ITC method for efficient characterization of bi-substrate enzyme kinetics.
  • To enable rapid mapping of reaction velocity as a function of two substrate concentrations.
  • To overcome limitations of current ITC techniques for enzyme kinetic studies.

Main Methods:

  • Development of a 2D-ITC technique for simultaneous kinetic data acquisition.
  • Performing a single experiment lasting approximately 2 hours.
  • Application of 2D-ITC to rabbit muscle pyruvate kinase (rMPK) for proof-of-principle.

Main Results:

  • The 2D-ITC method generated approximately 7000 catalytic rate measurements in a single experiment.
  • It accurately determined the random sequential mechanism of rMPK.
  • Identified allosteric regulation by phenylalanine (Phe), including alleviating ATP product inhibition, a novel finding.

Conclusions:

  • 2D-ITC offers a highly efficient and detailed approach for enzyme kinetic characterization.
  • The method provides insights comparable to or exceeding existing assays.
  • The findings on rMPK allosteric regulation by Phe have significant implications for cellular enzyme activity regulation.