Characterization and kinetic mechanism of catalytic domain of human vascular endothelial growth factor receptor-2 tyrosine kinase (VEGFR2 TK), a key enzyme in angiogenesis.

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.

Biochemical role of the Cryptococcus neoformans ADE2 protein in fungal de novo purine biosynthesis.

Comparative studies of 5-aminoimidazole ribonucleotide (AIR) carboxylases from Escherichia coli and Gallus gallus have identified this central step in de novo purine biosynthesis as a case for unusual divergence in primary metabolism. Recent discoveries establish the fungal AIR carboxylase, encoded by the ADE2 gene, as essential for virulence in certain pathogenic organisms. This investigation is a biochemical analysis that links the fungal ADE2 protein to the function of the E. coli AIR carboxylase system. A cDNA clone of ADE2 from Cryptococcus neoformans was isolated by genetic complementation of a purE-deficient strain of E. coli. High-level expression of the C. neoformans ADE2 was achieved, which enabled the production and purification of AIR carboxylase. Amino acid sequence alignments, C-terminal deletion mutants, and biochemical assays indicate that the ADE2 enzyme is a two-domain, bifunctional protein. The N-terminal domain is related to E. coli PurK and a series of kinetic experiments show that the ADE2-PurK activity uses AIR, ATP, and HCO3- as substrates. The biosynthetic product of the ADE2-PurK reaction was identified as N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) by 1H NMR, thus confirming that the C-terminal domain contains a catalytic activity similar to that of the E. coli PurE. By using an in situ system for substrate production, the steady-state kinetic constants for turnover of N5-CAIR by ADE2 were determined and together with stoichiometry measurements, these data indicate that ADE2 has a balance in the respective catalytic turnovers to ensure efficient flux. Distinctive features of the PurE active site were probed using 4-nitro-5-aminoimidazole ribonucleotide (NAIR), an analog of the product 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). NAIR was shown to be a selective inhibitor of the ADE2-PurE activity (K1 = 2.4 microM), whereas it is a slow-binding inhibitor of the G. gallus enzyme which further distinguishes the fungal ADE2 from the G. gallus AIR carboxylase. As such, this enzyme represents a novel intracellular target for the discovery of antifungal agents.