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-35S]GTP[S]/mol of protein with a Kd value of 56.83 nM. The ratios k(off GDP)/k(off GTP) 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 Pi release against [gamma-32P]GTP bound ORrab2 in a steady state and the rate of hydrolysis of [gamma-32P]GTP (kGTPase) were calculated to be 432 x 10(-4) +/- 8 x 10(-4) min(-1) and 172 x 10(-4) +/- 2 x 10(-4) min(-1), respectively, in the presence of 0.1% C12E10 and 1 mM MgSO4.
Seo
H S
HS
Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju, Korea.
Choi
C H
CH
Kim
H Y
HY
Jeong
J Y
JY
Lee
S Y
SY
Cho
M J
MJ
Bahk
J D
JD
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D012275:Oryza / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020691:rab GTP-Binding Proteins
D006150:Guanine Nucleotides; D010940:Plant Proteins; D011994:Recombinant Proteins; C109047:rab2 protein, Oryza sativa; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020691:rab GTP-Binding Proteins