Interaction of 6-thioguanylic acid homologues with ribonuclease T 1 Spectrophotometric and gel filtration studies

Abstract

The interaction between ribonuclease T 1 (RNAase T 1) (EC 2.7.7.26) and 6-thioguanylic acid homologues, characterized by an absorption band centered at 342 nm, was compared with that between RNAase T 1 and guanylic acids. The difference absorption spectra of the complexes of RNAase T 1 with thioguanosine 2′(3′)-phosphate, thioguanosine 2′(3′),5′-diphosphate and thioguanosine 2′,3′-cyclic phosphate 5′-phosphate were similar in shape to those of the complexes with guanosine 2′(3′)-phosphate and guanosine 2′(3′),5′-diphosphate, but were at about 60–80 nm longer wavelengths, with maxima at 326 and 351 nm at pH 5.6. The gel filtration studies demonstrated that stoichiometric complexes were formed between the enzyme and these thioguanylic acids. These results seem to indicate that the modes of interaction of RNAase T 1 with these thionucleotides are similar to those with guanylic acids. The circular dichroism band (negative) of thioguanosine 2′(3′),5′-diphosphate around 342 nm increased its intensity when it was complexed with RNAase T 1, suggesting conformational changes in the nucleotide and/or interaction between the base moiety of the nucleotide and chromophores of the enzyme. The order of the affinity of guanosine and thioguanosine derivatives for RNAase T 1, estimated from spectrophotometric and gel filtration studies, was as follows: guanosine 2′(3′)-phosphate, guanosine 2′(3′),5′-diphosphate > thioguanosine 2′(3′)-phosphate, thioguanosine 2′(3′),5′-diphosphate, thioguanosine 2′,3′-cyclic phosphate 5′-phosphate > thioguanosine 5′-phosphate > thioguanosine 2′,3′-cyclic phosphate, thioguanosine. The present data suggest that the enzyme possesses a subsite which is responsible for specific interaction with the 5′-phosphoryl groups of substrates or substrate analogues.