D. Kukla

Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor: II. Crystallographic refinement at 1.9 Å resolution☆

Abstract The crystal structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor has been refined with data to 1.9 A resolution, using a procedure described by Deisenhofer & Steigemann (1974) in their refinement of the crystal structure of the free inhibitor. This procedure involves cycles consisting of phase calculation using the current atomic model, Fourier synthesis using these phases and the observed structure factor amplitudes and Diamonds real-space refinement (Diamond, 1971,1974). At various stages, difference Fourier syntheses are calculated to detect and correct gross errors in the model and to localize solvent molecules. The refinement progressed smoothly, starting with the model obtained from the isomorphous Fourier map at 2.6 A resolution. The R -factor is 0.23 for 20,500 significantly measured reflections to 1.9 A resolution, using an over-all temperature factor of 20 A 2 . The estimated standard deviation of atomic positions is 0.09 A. An objective assessment of the upper limit of the error in the atomic coordinates of the final model is possible by comparing the inhibitor component in the model of the complex with the refined structure of the free inhibitor (Deisenhofer & Steigemann, 1974). The mean deviation of main-chain atoms of the two molecular models in internal segments is 0.25 A, of main-chain dihedral angles 5.1 ° and side-chain dihedral angles 6.5 °. A comparison of the trypsin component with α-chymotrypsin (Birktoft & Blow, 1972) showed a mean deviation of main-chain atoms of 0.75 A. The structures are closely similar and the various deletions and insertions cause local structural differences only.

Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor: Crystal structure determination and stereochemistry of the contact region

Abstract The structure of the complex of bovine trypsin and bovine pancreatic trypsin inhibitor has been determined by crystal structure analysis at 2.8 A resolution. The structure is closely similar to the model predicted from the structures of the components. The complex is a tetrahedral adduct with a covalent bond between the carbonyl carbon of Lys-15I of the inhibitor and the γ-oxygen of Ser-195 of the enzyme. The imidazole of His-57 is hydrogen-bonded to Asp-102 and the bound seryl γ-oxygen in accord with the histidine being charged. The negatively charged carbonyl oxygen of Lys-15I forms two hydrogen bonds with the amide nitrogens of Gly-193 and Ser-195. Protonation of the leaving group N-H of Ala-16I to form an acyl-complex requires a conformational change of the imidazole of His-57. The tetrahedral adduct is further stabilized by hydrogen bonds between groups at the leaving group side and inhibitor and enzyme, which would be weakened in the acyl-enzyme. The kinetic data of inhibitor-enzyme interaction are reconciled with the structural model, and relations between enzyme-inhibitor interaction and productive enzyme-substrate interaction are proposed.

The basic trypsin inhibitor of bovine pancreas

The basic pancreatic trypsin inhibitor was first described by Kunitz and Northrop ~t]. I t binds to and inhibits trypsin with a binding constant of approximately t013 (M -I) at alkaline pH [21. I t also inhibits e-cliymotrypsin, kallikrein, and plasmin [31. I t has a molecular weight of 6,500 and consists of 58 amino acids. The amino acid sequence has been determined by Kassell and Laskowski [4] and by Anderer and H6rnle ES] (Fig. 1). I t contains three disulfide bridges and has a large excess of basic residues over acidic amino acids.

The structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor

The structure of the complex between anhydro-trypsin and pancreatic trypsin inhibitor has been determined by difference Fourier techniques using phases obtained from the native complex (Huber et al., 1974). It was refined independently by constrained crystallographic refinement at 1.9 å resolution. The anhydro-complex has Ser 195 converted to dehydro-alanine. There were no other significant structural changes. In particular, the high degree of pyramidalization of the C atom of Lys 15 (I) of the inhibitor component observed in the native complex is maintained in the anhydro-species.