The refined crystal structure of a fully active semisynthetic ribonuclease at 1.8-A resolution.

Ribonuclease A

“…264 These data are difficult to interpret, however, because the threedimensional structure D121N semisynthetic variant deviates from that of RNase(1-118)‚ (111)(112)(113)(114)(115)(116)(117)(118)(119)(120)(121)(122)(123)(124). [265][266][267] Site-directed mutagenesis has been used to replace Asp121 with glutamate, asparagine, and alanine residues. 36,153,[268][269][270] The glutamate variant has approximately 17% of the activity of the wild-type enzyme for C>p hydrolysis.…”

“…The D121N semisynthetic variant has approximately 5% of the catalytic activity of the analogous wild-type semisynthetic enzyme . These data are difficult to interpret, however, because the three-dimensional structure D121N semisynthetic variant deviates from that of RNase(1−118)·(111−124). − …”

Structure (1.3 Å) and Charge States of a Ribonuclease A−Uridine Vanadate Complex: Implications for the Phosphate Ester Hydrolysis Mechanism

“…The structures of several ribonuclease complexes, including those containing inhibitors and nucleotide analogues, have also been determined, − leading to a further understanding of the structural features essential for substrate binding and enzyme catalysis. In addition, structural investigations of semisynthetic variants of ribonuclease have been carried out to address specific questions concerning catalysis and how the change of a specific amino acid residue can influence the structure. − Collectively, the structural work on RNase A has provided valuable insight into many aspects of the function of this enzyme, including many qualitative features of the catalytic mechanism. Shown in Figure is a three-dimensional ribbon representation of the C α backbone of UV−RNase A complex with the UV (uridine vanadate) and important active site residues, Gln11, His12, Lys41, and His119, identified.…”

NMR study of the positions of His-12 and His-119 in the ribonuclease A-uridine vanadate complex

“…As a consequence, does not hydrogen-bond to Asp-121 (Palmer et al, 1984), and the distance from the imidazole ring of His-119 to both the active site of the enzyme and the side chain of His-12 is several Angstroms greater than is seen in position A (Martin et al, 1987). In crystals of the fully active semisynthetic ribonuclease A (Martin et al, 1987) and of the Asn-121-and Ala-121-substituted semisynthetic ribonuclease analogs (de-Mel et al, 1992) that were grown in ammonium sulfate, pH 5.2, His-119 occupies primarily position B.…”

“…The observed differences in position A and B of His-119 of RNase A might be a function of differences in crystallographic and solution structures; however, they might be a result of differences in experimental conditions. The RNase A that exhibits His-119 primarily in the A position (Borkakoti et al, 1982;) was crystallized from alcohols, such as 40-60% ethanol at pH 5.2-5.7, and 43% t-butyl alcohol at pH 5.3; in contrast, RNase crystallized from salt solutions such as ammonium sulfate at pH 5.2 (Martin et al, 1987; deMel et al, 1992) exhibits His-119 in the B position. Moreover, NOE spectra of the RNase A/phosphate complex in aqueous solution in 0.2 M NaCl at pH 4.0 show His-119 predominantly near Val-118, which abuts the B position (Santoro et al, 1993).…”