Crystals of a catalytically defective, semisynthetic ribonuclease isomorphous with those of the fully active parent enzyme

Structural investigation of catalytically modified F120L and F120Y semisynthetic ribonucleases

“…Crystals of RNase 1-118:lll-124 and the two analogs were all grown from ammonium sulfate/cesium chloride salt solutions buffered at pH 5.2 as described earlier (Sasaki et al, 1979;Doscher et al, 1983a). CsCl was removed by transferring the crystals into a stabilizing buffer of 80% saturated ammonium sulfate, 0.1 M ammonium acetate, pH 5.2.…”

“…Conversely, the subsequent replacement of Phe 120 by tyrosine in RNase 1-1 18: 11 1-124 resulted in an enzyme with full activity toward RNA and cytidine 2',3'-cyclic phosphate and enhanced activity toward uridine 2',3'-cyclic phosphate (Hodges & Merrifield, 1974). In order to understand more completely the way in which the structure at position 120 modulates catalytic efficiency in this enzyme, we have now determined the 2.0-A structures of both F120L (R = 0.161) and F120Y ( R = 0.184) using crystals grown under conditions identical to those used to crystallize (Doscher et al, 1983a). Crystals of both analogs proved to be in the same space group and to have very nearly the same unit cell dimensions as the fully active parent complex (Martin et al, 1987; a References: 'Herries et al, 1962;'Rondaet al, 1976;3deMel et al, 1992;4Lin et al, 1972. Assay conditions: 0.20 M KCI, pH maintained at 6.00 with a pHstat, 25 "C, 0.92-8.8 mM substrate, RNase concentration not given.…”

Ribonuclease A

“…These studies were successful in illuminating molecular aspects of enzymatic catalysis, protein−protein interactions, and protein−nucleic acid interactions, and were the harbinger of current work on proteins containing variant or nonnatural amino acid residues. Work on the structure and function of another semisynthetic ribonuclease, RNase-(1−118)·(111−124), , has also made significant contributions. ,,,,− Recently, the RNase S system has spawned or at least facilitated the development of many innovative technologies.…”