cyclic-gmp and guanosine-2--3--cyclophosphorothioate

The backbone dynamics of RNase T1 in the presence of exo-guanosine 2',3'-cyclophosphorothioate (exo-cGPS isomer), which is a productive substrate, and in the presence of 3'-guanylic acid (3'GMP), which is an nonproductive substrate, were examined using (15)N nuclear magnetic resonance. Although the X-ray crystal structure suggests that the modes of binding of these substrates to the active-site cleft are very similar, the order parameters in a number of regions in RNase T1 complexed with exo-cGPS isomer were different from those with 3'GMP. Moreover, the chemical exchange in line width observed for RNase T1 complexed with exo-cGPS isomer was also different from that observed for RNase T1 complexed with 3'GMP. From these results, we concluded that the internal motions in RNase T1 complexed with a productive substrate were not always identical to those in RNase T1 complexed with a nonproductive substrate.

Topics: Cyclic GMP; Guanosine Monophosphate; Models, Molecular; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular; Ribonuclease T1; Thionucleotides

Here we present a time-resolved crystallographic analysis of the hydrolysis of exo (Sp) guanosine 2',3'-cyclophosphorothioate by RNase T1. The use of a slow substrate and fast crystallization methods made it possible to perform the study with conventional data-collection techniques. The results support the idea that the hydrolysis reaction proceeds through a mechanism that is the inverse of the transesterification reaction. In addition, the structures provide an explanation for the differential behavior of RNase T1 towards exo- and endo-cyclic thiophosphates.

Topics: Aspergillus oryzae; Binding Sites; Computer Simulation; Crystallography, X-Ray; Cyclic GMP; Hydrolysis; Models, Molecular; Molecular Conformation; Molecular Sequence Data; Protein Conformation; Ribonuclease T1; Substrate Specificity; Thionucleotides; Time Factors

We used mutants of RNase T1 and the Rp isomer of a thiosubstituted substrate to determine stereospecific thioeffects on catalysis. The analysis reveals subtle structural and functional changes in the intermolecular transition state interactions. Tyr 38 contributes to catalysis through a hydrogen bond with the pro-Rp oxygen. Y38F RNase T1 prefers the Rp thiosubstituted analog over the natural phosphodiester substrate that is favored by the wild type enzyme.

Topics: Amino Acid Substitution; Catalysis; Cyclic GMP; Models, Molecular; Mutagenesis, Site-Directed; Protein Engineering; Ribonucleases; RNA; Substrate Specificity; Thionucleotides

A modified method for the synthesis and separation of endo and exo guanosine 2',3'-cyclophosphorothioate (cGPS) has been developed. The exo diastereoisomer has been co-crystallized with RNase T1. cGPS is known to be a RNase T1 inhibitor but is also a very slow substrate. It was hydrolyzed during the crystallization, leaving 3'-guanylic acid (3'-GMP) in the active site. As a guanosine was also found to be bound in a subsite, the enzyme contains the products of the reaction of guanylyl-3',5'-guanosine. The structure was refined to a resolution of 1.7 A and yielded a final R value of 14.5%. In contrast to previous 3'-GMP complexes of RNase T1, the ribose phosphate moiety of the inhibitor is in contact with all the active site residues. The phosphate forms hydrogen bonds with Asn36, Tyr38, Arg77, His92, and with Asn49 from a symmetry-related molecule. The ribose 2'-OH is hydrogen-bonded to both Glu58 and His40. The interactions in the active site of the present structure are compared to those found in the 2'-GMP complex of RNase T1.

Topics: Binding Sites; Crystallography, X-Ray; Cyclic GMP; Guanosine; Guanosine Monophosphate; Hydrolysis; Molecular Structure; Ribonuclease T1; Stereoisomerism; Thionucleotides

The structure of the complex of ribonuclease from Streptomyces aureofaciens (RNase Sa) with exo guanosine 2',3'-cyclophosphorothioate has been refined against 0.2-nm resolution synchrotron data using, as a starting model, coordinates from the RNase Sa: 2'-GMP complex. The refinement was based on all data over 1.0-0.2 nm and converged to a crystallographic R factor of 11.9%. This is the first structure of a microbial ribonuclease complexed with a 2',3'-cyclophosphorothioate, which is a thio analogue of the intermediate of the two-step reaction. However, exo guanosine 2',3'-cyclophosphorothioate is bound in a non-functional mode and is not hydrolysed. This structure therefore does not provide direct evidence on the identity of the amino acid residues responsible for catalytic cleavage of the substrate. However, based on present and previous results, a plausible model is proposed for the complex of the cyclic intermediate which acts as substrate for the second step of the catalysis.

Topics: Binding Sites; Crystallization; Cyclic GMP; Guanosine Monophosphate; Hydrolysis; Ribonucleases; Streptomyces aureofaciens; Temperature; Thionucleotides; X-Ray Diffraction