1H-NMR investigation of the interaction between RNase T1 and a novel substrate analog, 2′-deoxy-2′-fluoroguanylyl-(3′–5′)uridine

AbstractThe interaction between RNase T1 and a non-hydrolysable substrate analog, 2′-deoxy-2′-fluoroguanylyl-(3′–5′)uridine (GfpU), was investigated using 1H-NMR spectroscopy. In the complex, the Gfp portion takes the syn form around the glycosidic bond and the 3′-endo form for the ribose moiety, similar to those found in 3′-GMP and 2′-deoxy-2′-fluoroguanosine 3′-monophosphate (Gfp). However, in contrast to the cases of these two inhibitors, the complex formation with GfpU at pH 6.0 was found to shift the His-40 C2 proton resonance of RNase T1 to high field as much as 1 ppm. At pH 6.0, this histidine residue appears to be unprotonated in the complex, but is protonated in the free enzyme (pKa of His-40 being 7.9). His-40, rather than Glu-58, is probably involved in the catalytic mechanism as a Lewis base, supporting the recent results from site-directed mutagenesis

ENGINEERING OF RNASE T 1

Ribonuclease Tl (RNase T1) was found in 1975 by Sato and Egamil from Aspergillus oryzae. This enzyme catalyzes hydrolysis of single-stranded RNA at guanylic acid 3'- phosphodiester sites. By this property RNase Tl is extremely useful for elucidation of primary structure of RNA's together with pancreatic RNase. The amino acid sequence of RNase Tl has been reported in 1965 , but it was corrected later in 1985.2 This enzyme consists of 104 amino acids and two disulfide bridges between Cys 2 and 10, as well as Cys6 and 103. RNase T1 is very stable towards heating and acidic conditions and suitable for biochemical and physicochemical studies.3 Recently three dimensional structure of this enzyme was elucidated by X-ray crystallography4:5 as a complex with an inhibitor guanosine 2'-phosphate. In this paper we attempted to obtain some details of structure-function relationship of RNase Tl by means of protein engineering