Subject(s)
Ribonuclease, Pancreatic , Chemistry Techniques, Analytical/methods , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Fluorescent Dyes , In Vitro Techniques , Models, Molecular , Protein Conformation , Ribonuclease, Pancreatic/chemistry , Ribonuclease, Pancreatic/metabolismABSTRACT
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 approximately dArXdAdA approximately 6-carboxytetramethylrhodamine (6-FAM approximately dArXdAdA approximately 6-TAMRA), where X = C, U, A, or G. Wild-type RNase A cleaves 10(6)-fold faster when X = C than when X = A. Likewise, its affinity for the non-hydrolyzable oligonucleotide 6-FAM approximately d(CAA) is 50-fold greater than for 6-FAM approximately d(AAA). T45G RNase A cleaves 6-FAM approximately dArAdAdA approximately 6-TAMRA 10(2)-fold faster than does the wild-type enzyme. The structure of crystalline T45G RNase A, determined at 1.8-A 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 approximately d(AAA). The importance of pentofuranosyl ring conformation to nucleotide specificity was probed with 6-FAM approximately d(AU(F)AA), where U(F) is 2'-deoxy-2'-fluorouridine. The conformation of the pentofuranosyl ring in dU(F) is known to be more similar to that in rU than dU. The affinity of wild-type RNase A for 6-FAM approximately d(AU(F)AA) is 50-fold lower than for 6-FAM approximately d(AUAA). This discrimination is lost in the T45G enzyme. Together, these data indicate that the side chain of Thr45 plays multiple roles-interacting 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.
Subject(s)
Pyrimidine Nucleotides/chemistry , Ribonuclease, Pancreatic/chemistry , Animals , Binding Sites/genetics , Carbohydrate Conformation , Catalysis , Cattle , Crystallization , Crystallography, X-Ray , Cytosine Nucleotides/chemistry , Cytosine Nucleotides/genetics , Glycine/genetics , Mutagenesis, Site-Directed , Purine Nucleotides/chemistry , Purine Nucleotides/genetics , Pyrimidine Nucleotides/genetics , Ribonuclease, Pancreatic/genetics , Ribonucleosides/chemistry , Ribonucleosides/genetics , Substrate Specificity/genetics , Threonine/genetics , Uracil Nucleotides/chemistry , Uracil Nucleotides/geneticsABSTRACT
A substrate for a hypersensitive assay of ribonucleolytic activity was developed in a systematic manner. This substrate is based on the fluorescence quenching of fluorescein held in proximity to rhodamine by a single ribonucleotide embedded within a series of deoxynucleotides. When the substrate is cleaved, the fluorescence of fluorescein is manifested. The optimal substrate is a tetranucleotide with a 5',6-carboxyfluorescein label (6-FAM) and a 3',6-carboxy-tetramethylrhodamine (6-TAMRA) label: 6-FAM-dArUdAdA-6-TAMRA. The fluorescence of this substrate increases 180-fold upon cleavage. Bovine pancreatic ribonuclease A (RNase A) cleaves this substrate with a k (cat)/ K (m)of 3.6 x 10(7)M(-1)s(-1). Human angiogenin, which is a homolog of RNase A that promotes neovascularization, cleaves this substrate with a k (cat)/ K (m)of 3. 3 x 10(2)M(-1)s(-1). This value is >10-fold larger than that for other known substrates of angio-genin. With these attributes, 6-FAM-dArUdAdA-6-TAMRA is the most sensitive known substrate for detecting ribo-nucleolytic activity. This high sensitivity enables a simple protocol for the rapid determination of the inhibition constant ( K (i)) for competitive inhibitors such as uridine 3'-phosphate and adenosine 5'-diphos-phate.
Subject(s)
Fluoresceins/metabolism , Proteins/metabolism , Rhodamines/metabolism , Ribonuclease, Pancreatic/metabolism , Adenosine Diphosphate/metabolism , Adenosine Diphosphate/pharmacology , Animals , Cattle , Deoxyadenosines/chemistry , Deoxyadenosines/metabolism , Fluoresceins/chemistry , Fluorescence , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Humans , Kinetics , Proteins/antagonists & inhibitors , Pyrimidines/chemistry , Pyrimidines/metabolism , Rhodamines/chemistry , Ribonuclease, Pancreatic/antagonists & inhibitors , Sensitivity and Specificity , Thermodynamics , Uridine Monophosphate/metabolism , Uridine Monophosphate/pharmacologyABSTRACT
Bovine pancreatic ribonuclease A (RNase A) is a distributive endoribonuclease that catalyzes the cleavage of the P-O5' bond of RNA on the 3' side of pyrimidine residues. Here, RNase A is shown to cleave the P-O5' bond of a pyrimidine ribonucleotide faster when the substrate is embedded within a longer tract of poly(adenylic acid) [poly(A)] or poly(deoxyadenylic acid) [poly(dA)]. These data indicate that a ribonuclease can diffuse in one dimension along a single-stranded nucleic acid. This facilitated diffusion is mediated by Coulombic interactions, as the extent is diminished by the addition of NaCl. RNase A is more effective at cleaving a pyrimidine ribonucleotide embedded within a poly(dA) tract than within a poly(deoxycytidylic acid) [poly(dC)] tract. T45G RNase A, which catalyzes the processive cleavage of poly(A) but the distributive cleavage of poly(cytidylic acid) [poly(C)], has the same preference. Apparently, processive catalysis by the T45G enzyme arises from the expanded substrate specificity of the variant superimposed upon an intrinsic ability to diffuse along poly(A). Homologous ribonucleases with cytotoxic activity may rely on facilitated diffusion along poly(A) tails for efficient degradation of the essential information encoded by cellular mRNA.
