Guanine-nucleotide binding and hydrolyzing kinetics of ORrab2, a rice small GTP binding protein expressed in Escherichia coli

The ORrab2 gene encodes a GTP-binding protein of 23.169 kDa. The deduced amino acid sequence shows that ORrab2 has the motifs conserved among small GTP-binding proteins in plants and that it shares sequence identity with Atrab2 (93.0%), Hrab2 (85.2%), Hrab4 (51.9%), Hrab1 (46.2%), YPT (40.7%), Hrab3B (40.0%), Hrab3A (38.1%), SEC4 (38.1%), Hrab5 (34.3%) and Hrab6 (32.4%). To analyze the biochemical properties of this protein, an ORrab2 cDNA was overexpressed in Escherichia coli and the protein purified by Ni2+-nitrilotriacetic acid agarose and hydroxyapatite column chromatography. The molecular mass of the protein bearing a His-tag is approximately 28.2 kDa. The guanine-nucleotide binding and hydrolyzing activity of ORrab2 increased with non-ionic C12E10 (polyoxyethylene 10-lauryl ether) and ionic Chaps detergent treatment. ORrab2 bound maximally 1.03 mol of [gamma-S-35]GTP[S]/mol of protein with a K-d value of 56.83 nM. The ratios k(offGDP)/k(offGTP) of ORrab2 were 3.63 for the control, 3.7 in the presence of C12E10, and 3.83 with Chaps, indicating that ORrab2 has a higher affinity for GTP than GDP. The rate (k(cat)) of P-i release against [gamma-P-32]GTP bound ORrab2 in a steady state and the rate of hydrolysis of [gamma-P-32]GTP (k(GTPase)) were calculated to be 432X10(-4)+/-8X10(-4) min(-1) and 172X10(-4)+/-2X10(-4) min(-1), respectively, in the presence of 0.1% C12E10 and 1 mM MgSO4.