X-RAY CRYSTALLOGRAPHIC AND CALORIMETRIC STUDIES OF THE EFFECTS OF THEMUTATION TRP59-]TYR IN RIBONUCLEASE-T1

Two mutants of ribonuclease T1 (RNaseT1), [59-tyrosine]ribonuclease T1 (W59Y) and 1]45-tryptophan,5 9-tyrosine] ribonuclease T1 (Y45W/W59Y) possess between 150 % and 190 % wild-type activity. They have been cry stallised as complexes of the inhibitor 2'-guanylic acid and analysed by X-ray diffraction at resolutions of 0.23 nm and 0.24 nm, respective ly. The space group for both is monoclinic, P2(1), with two molecules/ asymmetric unit, W59Y: a = 4.934 nm, b = 4.820, c = 4.025 nm, beta = 9 0.29 degrees. Y45W/W59Y: a = 4.915 nm, b = 4.815 nm, c = 4.015 nm, bet a = 90.35 degrees. Compared to wild-type RNaseT1 in complex with 2'-gu anylic acid (2'GMP) both mutant inhibitor complexes indicate that the replacement of Trp59 by Tyr leads to a 0.04-nm inward shift of the sin gle a-helix and to significant differences in the active-site geometry , inhibitor conformation and inhibitor binding. Calorimetric studies o f a range of mutants [24-tryptophan]ribonuclease T1 (Y24W), [42-trypto phan]ribonuclease T1 (Y42W) [45-tryptophan]ribonuclease T1 (Y45W), [92 -alanine]ribonuclease T1 (H92A) and [92-threonine]ribonuclease T1 (H92 T) with and without the further mutation Trp59-->Tyr showed that mutan t proteins for which Trp59 is replaced by Tyr exhibit slightly decreas ed thermal stability.