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.

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