The initial proteolysis of native bovine pancreatic ribonuclease A by subtilisin BPN' (Nagarse) occurs at either the bond between residues 21 and 22 or the previously established position between residues 20 and 21. As a result, the ribonuclease S produced contains a mixture of S-peptide molecules composed of

A senisynthetic RNase, RNase-(1-118)-(111-124), consising of a noncovalent complex between residues 1-118 of RNase (obtained from the proteolytic digestion of RNase A), and a synthetic 14-residue peptide containing residues 111-124 of RNase, exhibits 98% of the enzymatic activity of bovine pancreatic ribonuclease A (EC 3.1.27.5). The replacement of aspartic acid-121 by asparagine in this semisynthetic RNase to form the "D121N" analog reduces kd,/K. to 2.7% of the value for RNase A. In the present work, lH NMR spectroscopy has been used to probe the ionization sates of Pis12, His 9 , and His"' in this catalytically defective semisyn- combined with the corresponding peptide in which no amino acid changes have been introduced, the full enzymatic activity of RNase-(1-118)-(111-124) is generated (6). The overlap between the peptide and RNase-(1-118), at residues 111-118, is required to achieve both good binding and precise alignment of the two chains (7). A refined crystal structure at 1.8-A resolution of RNase-(1-118)-(111-124), the fully active parent complex, has been determined (8).The assignments of the C2 proton NMR resonances for each of the four histidines in bovine pancreatic RNase A and their pKa values have been made in several laboratories (9)(10)(11)(12)(13)

Structures of a semisynthetic RNase have been obtained to a resolution of 2.0 A at pH values of 5.2, 6.5, 7.5, and 8.8, respectively. The principle structural transformation occurring over this pH range is the conversion of the side chain of active site residue His-119 from one conformation (chi 1 = -43 degrees to -57 degrees) at low pH to another (chi 1 = +159 degrees to +168 degrees) at higher pH values. On the basis of this observation, a model is proposed that reconciles the disparate pK values for His-119 in the enzyme-substrate complex that have been deduced from kinetic studies and from proton NMR measurements in the presence of pseudosubstrates.

No abstract

No abstract

The fully active semisynthetic enzyme formed by the non-covalent interaction of residues l-l 18 of bovine pancreatic ribonuclease and a synthetic tetradecapeptide containing residues 111-124 of the enzyme has allowed a direct test of the role of aspartic acid-121 in the functioning of the molecule. Replacement of this residue by asparagine results in a derivative that is 4.5% active against cytidine 2' ,3'-cyclic phosphate at pH 7.0 under standard assay conditions. Further studies with the same substrate at pH 5.8 reveal that the reduced activity results entirely from a diminished catalytic efficiency and not from a decreased affinity for substrate. Bovine pancreatic ribonucleaseActive site

No abstract

The proton magnetic resonance spectrum at 300 MHz of the histidine residues in a semisynthetic derivative of bovine pancreatic ribonuclease (RNase A) has been determined. The derivative RNase 1-118 . 111-124 was prepared by enzymically removing six residues from the COOH terminus of the protein (positions 119-124) and then complementing the inactive RNase 1-118 with a chemically synthesized peptide containing the COOH-terminal 14 residues of ribonuclease (RNase 111-124) [Lin, M.C., Gutte, B., Moore, S., & Merrifield, R.B. (1970) J. Biol. Chem. 245, 5169-5170]. Comparison of the line positions of the C(2)-1H resonances of these residues and of their pH dependence with those reported by other workers has allowed assignment of the resonances to individual residues, as well as the determination of individual pK values for histidine-12, histidine-105, and histidine-119. The assignment of histidine-119 was confirmed by the use of a selectively deuterated derivative. The titration behavior of all four histidine residues is indistinguishable from that observed by others for bovine pancreatic ribonuclease A. Partial dissociation of the noncovalent semisynthetic complex was evident at 30 degrees C, pH 4.0, 0.3 M NaCl; pertinent spectra were analyzed to provide an estimate of the association constant between the component chains under these conditions of 1.9 X 10(3) M-1.