Residues Involved in the Catalysis, Base Specificity, and Cytotoxicity of Ribonuclease from Rana catesbeianaBased upon Mutagenesis and X-ray Crystallography
| Autor: | Sui-Chi Wang, Imameddin Amiraslanov, You-Di Liao, Ying-Jen Leu, Yen-Chywan Liaw, Ya-Yun Hsiao, Shuenn-Shing Chern |
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| Rok vydání: | 2003 |
| Předmět: |
Models
Molecular Threonine DNA Complementary Guanine Cell Survival RNase P Stereochemistry Genetic Vectors Molecular Sequence Data Glutamic Acid Crystallography X-Ray Biochemistry Catalysis Mass Spectrometry Substrate Specificity chemistry.chemical_compound Ribonucleases Animals Humans Amino Acid Sequence Enzyme kinetics Ribonuclease Molecular Biology Rana catesbeiana Sequence Homology Amino Acid biology Hydrogen bond Circular Dichroism Lysine Mutagenesis RNA Ribonuclease Pancreatic Cell Biology Recombinant Proteins Protein Structure Tertiary Kinetics chemistry Mutation biology.protein Cytosine Hydrogen |
| Zdroj: | Journal of Biological Chemistry. 278:7300-7309 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m206701200 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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