Characterization and Kinetic Mechanism of Catalytic Domain of Human Vascular Endothelial Growth Factor Receptor-2 Tyrosine Kinase (VEGFR2 TK), a Key Enzyme in Angiogenesis

• Published in: Biochemistry (Easton) Vol. 37; no. 47; pp. 16788 - 16801
• Main Authors: Parast, Camran V, Mroczkowski, Barbara, Pinko, Chris, Misialek, Shawn, Khambatta, Godrej, Appelt, Krzysztof
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 24.11.1998
• Subjects: Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - metabolism; Binding Sites; Binding, Competitive; Cadmium - pharmacology; Catalysis; Enzyme Activation; Humans; Kinetics; Magnesium - metabolism; Manganese - metabolism; Neovascularization, Physiologic; Phosphorylation; Protein Structure, Tertiary; Receptor Protein-Tyrosine Kinases - antagonists & inhibitors; Receptor Protein-Tyrosine Kinases - chemistry; Receptor Protein-Tyrosine Kinases - metabolism; Receptors, Growth Factor - antagonists & inhibitors; Receptors, Growth Factor - chemistry; Receptors, Growth Factor - metabolism; Receptors, Vascular Endothelial Growth Factor; Substrate Specificity; Time Factors
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi981291f

Vascular endothelial growth factor (VEGF) is a dimeric protein which induces formation of new blood vessels (angiogenesis) through binding to VEGF-receptor-2 tyrosine kinase (VEGFR2 TK) or KDR (kinase insert domain-containing receptor) on the surface of endothelial cells. Angiogenesis has been shown to be essential for malignancy of tumors; therefore, VEGFR2 TK is a potential therapeutic target for the treatment of cancer. Sequence homology studies indicate that VEGFR2 TK contains three domains:  extracellular (ligand-binding domain), transmembrane, and intracellular (catalytic domain). In this work, the catalytic domain of VEGFR2 TK was cloned and expressed in a soluble active form using a baculovirus expression system. In the absence of ligand, the enzyme is shown to catalyze its autophosphorylation in a time-dependent and enzyme-concentration-dependent manner, consistent with a trans mechanism for this reaction. Mass spectrometry analysis revealed incorporation of 5.5 ± 0.5 mol of phosphate/mole of enzyme (monomer). In addition, the enzyme was shown to catalyze phosphorylation of a synthetic peptide, poly(E4Y). Using poly(E4Y) as substrate, the kinetic constants of both native and phosphorylated enzyme were determined. Enzyme phosphorylation increased catalytic efficiency of the enzyme by at least an order of magnitude. Furthermore, the enzyme was shown to catalyze the reverse reaction using phospho-poly(E4Y) as substrate. Cd2+ was found to be an inhibitor of the enzyme. Kinetic studies revealed that inhibition by Cd2+ was competitive with respect to Mg2+ and noncompetitive with respect to MgATP. These results indicate that Cd2+ competes for a second metal-binding site. Therefore, the reaction catalyzed by this enzyme was treated as a terreactant system. The kinetic mechanism of VEGFR2 TK was elucidated through the use of steady-state kinetic studies. According to these studies, the enzyme binds Mg2+ and MgATP in a random fashion followed by ordered addition of the peptide substrate. The release of product is also ordered, with MgADP being released last. The order of substrate binding was confirmed by using AMP−PCP, a dead-end inhibitor.