The crystal structure of bovine seminal ribonuclease, a homodimeric enzyme closely related to pancreatic ribonuclease, has been refined at a nominal resolution of 1.9/k employing data collected on an electronic area detector. The final model consists of two chains containing 1990 non-H atoms, seven sulfate anions and 113 water molecules per asymmetric unit. The unit-cell parameters are a = 36.5 (1), b = 66.7 (1) and c = 107.5(2)~,, space group P22~2~. The R factor is 0.177 for 16 492 reflections in the resolution range 6.0-1.9 A and the deviations from ideal values of bond lengths and bond angles are 0.020 A and 3.7", respectively. The molecule is formed by two pancreatic like chains, which have their N-terminal segments interchanged so that each active site is formed by residues from both subunits. The two chains are related by a non-crystallographic twofold symmetry and are covalently linked by two consecutive disulfide bridges, which form an unusual sixteen-membered ring across the dimer interface. The deviations from the molecular symmetry, the hydration shell and the sulfate-binding sites are also discussed in relation to the known structure of the pancreatic enzyme.
The influence of pH and buffer concentration on the rate of the isomerization of Asp residues has been analysed using model aspartic acid-containing tetrapeptides, in the pH range 1.5-1 0 at 37 "C and p = 1 mol dm-3. The reaction involves the reversible formation of an amino-succinimide intermediate.Kinetic evidence indicates that of the various forms of the Asp-peptide in acid-base equilibrium, only one, having the carboxylic Asp side chain in the neutral state and the N-H peptide group next to the Asp residue in the deprotonated state, reacts at an appreciable rate. The reaction involves nucleophilic attack b y the nitrogen atom of the peptide bond o n the carbonyl carbon of the Asp side chain, giving a cyclic tetrahedral intermediate. A t p H values lower than about 3 the cyclization step is rate-determining, but at higher pH the rate-determining step is the general acid-catalysed departure of the leaving group. This change of rate-determining step, together with the ionization of the carboxylic side chain of the Asp residue, produces a bell-shaped curve in the pH-rate profile for the cyclization reactions.
At 37 "C and pH 7.4 the spontaneous deamidation of Gln-peptide proceeds prevalently via a glutarimide intermediate and gives a mixture of a-Glu-and y-Glu-peptide.
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