Contribution of Gln9 and Phe80 to substrate binding in ribonuclease MC1 from bitter gourd seeds.
Ribonuclease MC1 (RNase MC1) isolated from bitter gourd (Momordica charantia) seeds specifically cleaves phosphodiester bonds on the 5'-side of uridine. The crystal structures of RNase MC1 in complex with 2'-UMP or 3'-UMP reveal that Gln9, Asn71, Leu73, and Phe80 are involved in uridine binding by hydrogen bonding and hydrophobic interactions [Suzuki et al. (2000) Biochem. Biophys. Res. Commun. 275, 572-576]. To evaluate the contribution of Gln9 and Phe80 to uridine binding, Gln9 was replaced with Ala, Phe, Glu, or His, and Phe80 with Ala by site-directed mutagenesis. The kinetic properties of the resulting mutant enzymes were characterized using cytidylyl-3',5'-uridine (CpU) as a substrate. The mutant Q9A exhibited a 3.7-fold increased K(m) and 27.6-fold decreased k(cat), while three other mutations, Q9F, Q9E, and Q9H, predominantly affected the k(cat) value. Replacing Phe80 with Ala drastically reduced the catalytic efficiency (k(cat)/K(m)) with a minimum K(m) value equal to 8 mM. It was further found that the hydrolytic activities of the mutants toward cytidine-2',3'-cyclic monophosphate (cCMP) were reduced. These results demonstrate that Gln9 and Phe80 play essential roles not only in uridine binding but also in hydrolytic activity. Moreover, we produced double Ala substituted mutants at Gln9, Asn71, Leu73, and Phe80, and compared their kinetic properties with those of the corresponding single mutants. The results suggest that these four residues may contribute to uridine binding in a mutually independent manner.
Numata
T
T
Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581, Japan.
Kimura
M
M
D019943:Amino Acid Substitution; D001665:Binding Sites / Q000502:physiology; D003563:Cyclic CMP / Q000378:metabolism; D005973:Glutamine / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D006868:Hydrolysis; D007700:Kinetics; D029777:Momordica charantia / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism*; D012639:Seeds / Q000201:enzymology; D013379:Substrate Specificity; D013816:Thermodynamics; D014529:Uridine / Q000031:analogs & derivatives / Q000378:metabolism
D005973:Glutamine; D003563:Cyclic CMP; D010649:Phenylalanine; D012260:Ribonucleases; C413748:ribonuclease MC1; D014529:Uridine