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Electrostatic environment surrounding the activation loop phosphotyrosine in the oncoprotein v-Fps
B C Leon1, I Tsigelny, J A Adams
1Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0506, USA.
Biochemistry
|August 22, 2001
Summary
Autophosphorylation of v-Fps at Tyr-1073 enhances catalysis. Specific arginine residues (Arg-1042, Arg-1066) are crucial for this activation, with mutations impacting enzyme activity and conformational changes.
Area of Science:
- Biochemistry
- Enzymology
- Protein Kinase Research
Background:
- The oncoprotein v-Fps is a tyrosine kinase involved in cellular signaling.
- Autophosphorylation of v-Fps at Tyr-1073 is known to enhance its catalytic activity.
- Understanding the structural basis of v-Fps activation is crucial for targeted therapeutic development.
Purpose of the Study:
- To investigate the role of specific arginine residues (Arg-1042 and Arg-1066) in v-Fps autophosphorylation and catalytic activity.
- To elucidate the mechanism by which these arginines influence phosphoryl transfer, substrate binding, and metal ion coordination.
- To characterize the impact of mutations in these residues on enzyme kinetics and conformational dynamics.
Main Methods:
- Site-directed mutagenesis to create R1042A and R1066A mutants of v-Fps.
- Enzyme kinetics assays to measure turnover rates, substrate binding, and metal ion affinities.
- Solvent perturbation studies to analyze conformational changes and phosphoryl transfer steps.
- Structural modeling based on the insulin receptor to understand residue interactions.
Main Results:
- Mutation of Arg-1042 or Arg-1066 to alanine resulted in weakly phosphorylated enzymes, indicating their importance for maximal catalytic activity.
- The R1042A mutation significantly slowed the catalytic rate (50-fold) due to impaired conformational changes, while R1066A primarily affected the phosphoryl transfer step.
- Mutant R1066A exhibited altered affinities for the second Mg(2+) ion and a substrate mimic, suggesting Arg-1042 and Arg-1066 induce strain in the inactive enzyme.
- Autophosphorylation and subsequent catalytic improvements in wild-type v-Fps compensate for reduced metal and substrate binding affinities.
Conclusions:
- Arg-1042 and Arg-1066 play critical roles in the activation mechanism of v-Fps by influencing conformational dynamics and phosphoryl transfer.
- These residues appear to stabilize a strained, repressed state of the enzyme, which is overcome upon activation.
- The findings provide insights into the intricate regulation of tyrosine kinase activity and potential strategies for modulating v-Fps function.