Activation Mechanism and Steady State Kinetics of Bruton's Tyrosine Kinase.
From: Department of Biochemical Pharmacology, Roche Palo Alto LLC, CA 94304, USA.
The Journal of biological chemistry
- Publish Date: Mar 2007
- ISSN: 0021-9258
- Volume: 282
- Issue: 12
- Pages: 8768-76
- Medium: Print
- Language: English
- Citation (JAMA): Dinh Marie, Grunberger Dorit, Ho Hoangdung, et al. Activation Mechanism and Steady State Kinetics of Bruton's Tyrosine Kinase.. J. Biol. Chem. Mar 2007;282:8768-76
Abstract
Bruton’s tyrosine kinase (BTK) is a member of the Tec non-receptor tyrosine kinase family that is involved in regulating B cell proliferation. To better understand the enzymatic mechanism of the Tec family of kinases, the kinetics of BTK substrate phosphorylation were characterized using a radioactive enzyme assay. We first examined whether autophosphorylation regulates BTK activity. Western blotting with a phosphospecific antibody revealed that BTK rapidly autophosphorylates at Tyr(551) within its activation loop in vitro. Examination of a Y551F BTK mutant indicated that phosphorylation of Tyr(551) causes a 10-fold increase in BTK activity. We then proceeded to characterize the steady state kinetic mechanism of BTK. Varying the concentrations of ATP and S1 peptide (biotin-Aca-AAAEEIY-GEI-NH2) revealed that BTK employs a ternary complex mechanism with KmATP = 84 +/- 20 microM and KmS1 = 37 +/- 8 microM. Inhibition studies were also performed to examine the order of substrate binding. The inhibitors ADP and staurosporine were both found to be competitive with ATP and non-competitive with S1, indicating binding of ATP and S1 to BTK is either random or ordered with ATP binding first. Negative cooperativity was also found between the S1 and ATP binding sites. Unlike ATP site inhibitors, substrate analog inhibitors did not inhibit BTK at concentrations less than 1 mm, suggesting that BTK may employ a “substrate clamping” type of kinetic mechanism whereby the substrate Kd is weaker than Km. This investigation of BTK provides the first detailed kinetic characterization of a Tec family kinase.
Mesh Headings (Keywords): Adenosine Diphosphate, Adenosine Triphosphate, Binding Sites, Binding, Competitive, Enzyme Activation, Humans, Kinetics, Models, Chemical, Mutation, Peptides, Protein Binding, Protein-Tyrosine Kinases, Staurosporine, Substrate Specificity, Tyrosine
Check for Full Text / PubMed Unique Identifier (PMID): 17264076
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