Excavating an Active Site: The Nucleobase Specificity of Ribonuclease A

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Abstract
Ribonuclease A (RNase A) catalyzes the cleavage of RNA after pyrimidine nucleotides. When bound in the active site, the base of a pyrimidine nucleotide forms hydrogen bonds with the side chain of Thr45. Here, the role of Thr45 was probed by using the wild-type enzyme, its T45G variant, X-ray diffraction analysis, and synthetic oligonucleotides as ligands and substrates. Catalytic specificity was determined with the fluorogenic substrate: 6-carboxyfluorescein∼dArXdAdA∼6-carboxytetramethylrhodamine (6-FAM∼dArXdAdA∼6-TAMRA), where X = C, U, A, or G. Wild-type RNase A cleaves 106-fold faster when X = C than when X = A. Likewise, its affinity for the non-hydrolyzable oligonucleotide 6-FAM∼d(CAA) is 50-fold greater than for 6-FAM∼d(AAA). T45G RNase A cleaves 6-FAM∼dArAdAdA∼6-TAMRA 102-fold faster than does the wild-type enzyme. The structure of crystalline T45G RNase A, determined at 1.8-Å resolution by X-ray diffraction analysis, does not reveal new potential interactions with a nucleobase. Indeed, the two enzymes have a similar affinity for 6-FAM∼ d(AAA). The importance of pentofuranosyl ring conformation to nucleotide specificity was probed with 6-FAM∼d(AUFAA), where UF is 2‘-deoxy-2‘-fluorouridine. The conformation of the pentofuranosyl ring in dUF is known to be more similar to that in rU than dU. The affinity of wild-type RNase A for 6-FAM∼ d(AUFAA) is 50-fold lower than for 6-FAM∼d(AUAA). This discrimination is lost in the T45G enzyme. Together, these data indicate that the side chain of Thr45 plays multiple rolesinteracting favorably with pyrimidine nucleobases but unfavorably with purine nucleobases. Moreover, a ribose-like ring disfavors the interaction of Thr45 with a pyrimidine nucleobase, suggesting that Thr45 enhances catalysis by ground-state destabilization.

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