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Residues Involved in the Catalysis, Base Specificity, and Cytotoxicity of Ribonuclease from Rana catesbeianaBased upon Mutagenesis and X-ray Crystallography*

Y. Leu,S. Chern,Sui-Chi Wang,Y. Hsiao,I. Amiraslanov,Y. Liaw,Y. Liao

Published 2003 in Journal of Biological Chemistry

ABSTRACT

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,kcat /Km 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 Å resolution. Residues Lys-9, His-10, Lys-35, and His-103 interacted directly with catalytic phosphate at the P1site, 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 B1 base specificity. Interestingly, Pyr-1 along with Lys-95 and Glu-97 form four hydrogen bonds with guanine at B2 site and determine B2base specificity.

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