Insight into tyrosine phosphorylation in v-Fps using proton inventory techniques

Area of Science:

• Biochemistry
• Enzymology
• Protein Kinase Research

Background:

• v-Fps is a nonreceptor tyrosine protein kinase involved in cellular signaling.
• Understanding the catalytic mechanism of kinases, particularly the phosphoryl group transfer step, is crucial for drug development and disease research.
• Peptide substrates are commonly used to probe kinase activity and specificity.

Purpose of the Study:

• To elucidate the mechanism of phosphoryl group transfer in the kinase domain of v-Fps.
• To differentiate the kinetic control mechanisms for two distinct peptide substrates.
• To investigate the role of proton transfer and solvent effects in the catalytic process.

Main Methods:

• Proton inventory and viscosometric techniques were employed to analyze the kinase reaction.
• Kinetic parameters (kcat, Ks, KI) were determined for peptide substrates and an inhibitor peptide.
• Solvent isotope effects and proton inventory plots were analyzed to understand the transition state and proton transfer events.

Main Results:

• Two peptide substrates, peptide I and peptide II, bind to v-Fps with similar affinities but exhibit different rate-limiting steps.
• Phosphoryl group transfer is rate-limiting for peptide I, while both transfer and product release control kcat for peptide II.
• Significant solvent isotope effects and distinct proton inventory profiles (linear for peptide I, 'bowed' for peptide II) were observed, indicating differences in the transition state and proton transfer.

Conclusions:

• The study reveals distinct catalytic mechanisms for different peptide substrates of v-Fps.
• A single proton transfer is associated with phosphoryl group transfer for peptide I, while peptide II involves a more complex transition state.
• The findings suggest a mechanism involving an acid-base catalyst or a conformational change coupled with hydrogen bond disruption.