Residues involved in the catalysis, base specificity, and cytotoxicity of ribonuclease from Rana catesbeiana based upon mutagenesis and X-ray crystallography.
The Rana catesbeiana (bullfrog) ribonucleases, which belong to the RNase A superfamily, exert cytotoxicity toward tumor cells. RC-RNase, the most active among frog ribonucleases, has a unique base preference for pyrimidine-guanine rather than pyrimidine-adenine in RNase A. Residues of RC-RNase involved in base specificity and catalytic activity were determined by site-directed mutagenesis, k(cat)/K(m) analysis toward dinucleotides, and cleavage site analysis of RNA substrate. The results show that Pyr-1 (N-terminal pyroglutamate), Lys-9, and Asn-38 along with His-10, Lys-35, and His-103 are involved in catalytic activity, whereas Pyr-1, Thr-39, Thr-70, Lys-95, and Glu-97 are involved in base specificity. The cytotoxicity of RC-RNase is correlated, but not proportional to, its catalytic activity. The crystal structure of the RC-RNase.d(ACGA) complex was determined at 1.80 A resolution. Residues Lys-9, His-10, Lys-35, and His-103 interacted directly with catalytic phosphate at the P(1) site, and Lys-9 was stabilized by hydrogen bonds contributed by Pyr-1, Tyr-28, and Asn-38. Thr-70 acts as a hydrogen bond donor for cytosine through Thr-39 and determines B(1) base specificity. Interestingly, Pyr-1 along with Lys-95 and Glu-97 form four hydrogen bonds with guanine at B(2) site and determine B(2) base specificity.
Leu
Ying-Jen
YJ
Institute of Biomedical Sciences and the Institute of Molecular Biology, Academia Sinica, Taipei 115, and the Department of Life Science, National Tsing-Hua University, Hsin-Chu 300, Taiwan.
Chern
Shuenn-Shing
SS
Wang
Sui-Chi
SC
Hsiao
Ya-Yun
YY
Amiraslanov
Imameddin
I
Liaw
Yen-Chywan
YC
Liao
You-Di
YD
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002470:Cell Survival; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000378:metabolism; D005822:Genetic Vectors; D018698:Glutamic Acid / Q000737:chemistry; D006147:Guanine / Q000378:metabolism; D006801:Humans; D006859:Hydrogen / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D011892:Rana catesbeiana / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry
D018076:DNA, Complementary; D011994:Recombinant Proteins; D013912:Threonine; D018698:Glutamic Acid; D006147:Guanine; D012313:RNA; D006859:Hydrogen; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; D008239:Lysine