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Related Experiment Videos

Nucleotide release and associated conformational changes regulate function in the COOH-terminal Src kinase, Csk.

J Shaffer1, G Sun, J A Adams

  • 1Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0506, USA.

Biochemistry
|September 12, 2001
PubMed
Summary

The COOH-terminal Src kinase (Csk) enzyme rapidly phosphorylates substrates and releases products. However, the overall reaction rate is limited by the slow release of ADP, suggesting coupled conformational changes.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • The COOH-terminal Src kinase (Csk) is crucial for regulating cellular processes by phosphorylating Src family kinases.
  • Previous studies focused on Csk steady-state kinetics, leaving individual catalytic steps unexamined.

Purpose of the Study:

  • To elucidate the catalytic pathway of Csk by investigating individual substrate phosphorylation steps.
  • To understand active-site mechanisms governing Csk function.

Main Methods:

  • Pre-steady-state and transient-state kinetic analyses were employed.
  • Rapid quench flow and stopped-flow techniques were utilized.
  • Viscosometry was used to probe conformational changes.

Main Results:

Related Experiment Videos

  • Csk-mediated phosphorylation exhibits biphasic kinetics: a rapid burst phase followed by a slow linear phase.
  • The phosphoryl transfer step is fast (k3 ≥ 140 s⁻¹) and favorable (k3/k-3 ≥ 6).
  • The rate-limiting step in steady-state turnover is the slow dissociation of ADP, coupled with conformational changes.

Conclusions:

  • Csk efficiently transfers phosphate from ATP and rapidly releases the phosphopeptide product.
  • The catalytic cycle is primarily limited by the slow release of ADP, indicating a conformational bottleneck.