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Related Experiment Videos

Angiotensin converting enzyme: substrate inhibition.

J R Schullek1, I B Wilson

  • 1Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.

Peptides
|March 1, 1989
PubMed
Summary
This summary is machine-generated.

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HEPES buffer is inert for angiotensin converting enzyme (ACE) inhibition. Substrate cooperativity was observed with Hipp-His-Leu, revealing key kinetic parameters for ACE.

Area of Science:

  • Biochemistry
  • Enzymology
  • Pharmacology

Background:

  • Angiotensin converting enzyme (ACE) plays a crucial role in the renin-angiotensin system.
  • Various buffers, including phosphate, borate, and Tris, are known to inhibit ACE activity.
  • The inertness of HEPES buffer in ACE inhibition requires further investigation for accurate kinetic studies.

Purpose of the Study:

  • To investigate substrate inhibition kinetics of ACE in HEPES buffer.
  • To determine if cooperativity exists in substrate binding to ACE's active and inhibitory sites.
  • To accurately quantify ACE kinetic parameters and specific activity in HEPES buffer.

Main Methods:

  • Measurements of substrate inhibition were conducted in HEPES buffer at pH 7.0 and temperatures of 25°C and 37°C.

Related Experiment Videos

  • A novel equation for substrate inhibition was employed to assess binding cooperativity.
  • Enzyme concentration was determined by titration with lisinopril to calculate kcat and specific activity.

    • Main Results:

    • Substrate inhibition of ACE was observed to be marked and complete in HEPES buffer.
    • Cooperativity in substrate binding (Hipp-His-Leu) to ACE was confirmed.
    • Key kinetic parameters including Km (0.21 mM) and K* (0.65 mM) at 37°C were determined.

    Conclusions:

    • HEPES buffer is suitable for studying ACE kinetics due to its inertness.
    • ACE exhibits cooperativity in substrate binding, providing insights into enzyme mechanism.
    • The specific activity of ACE in HEPES buffer was calculated as 33.7 units/mg at 37°C.