Functional and structural roles of the Cys14-Cys38 disulfide of bovine pancreatic trypsin inhibitor

Abstract

The disulfide bond between Cys14 and Cys38 of bovine pancreatic trypsin inhibitor lies on the surface of the inhibitor and forms part of the protease-binding region. The functional properties of three variants lacking this disulfide, with one or both of the Cys residues replaced with Ser, were examined, and X-ray crystal structures of the complexes with bovine trypsin were determined and refined to the 1.58-A resolution limit. The crystal structure of the complex formed with the mutant with both Cys residues replaced was nearly identical with that of the complex containing the wild-type protein, with the Ser oxygen atoms positioned to replace the disulfide bond with a hydrogen bond. The two structures of the complexes with single replacements displayed small local perturbations with alternate conformations of the Ser side chains. Despite the absence of the disulfide bond, the crystallographic temperature factors show no evidence of increased flexibility in the complexes with the mutant inhibitors. All three of the variants were cleaved by trypsin more rapidly than the wild-type inhibitor, by as much as 10,000-fold, indicating that the covalent constraint normally imposed by the disulfide contributes to the remarkable resistance to hydrolysis displayed by the wild-type protein. The rates of hydrolysis display an unusual dependence on pH over the range of 3.5-8.0, decreasing at the more alkaline values, as compared with the increased hydrolysis rates for normal substrates under these conditions. These observations can be accounted for by a model for inhibition in which an acyl-enzyme intermediate forms at a significant rate but is rapidly converted back to the enzyme-inhibitor complex by nucleophilic attack by the newly created amino group. The model suggests that a lack of flexibility in the acyl-enzyme intermediate, rather than the enzyme-inhibitor complex, may be a key factor in the ability of bovine pancreatic trypsin inhibitor and similar inhibitors to resist hydrolysis.

Figure 1

Ribbon diagram representation of the complex formed between BPTI (blue) and bovine cationic trypsin (green). The sulfur atoms of the six disulfide-bonded Cys residues of BPTI are shown as yellow spheres, and the side-chain atoms of Lys16 and Tyr35 of BPTI are shown as sticks. The backbone ribbon representing Lys15 and Ala16 is colored black to identify the region of the scissile bond. Drawn from the atomic coordinates in entry 2FTL of the Protein Data Bank, using the computer program PyMOL (http://www.pymol.org).

Figure 2

Hydrolysis of BPTI variants at pH 7.8 monitored by HPLC separation. Each of the indicated BPTI variants was incubated with an equimolar concentration of bovine trypsin at pH 7.8 and 25 °C. Samples of the reaction mixtures were withdrawn at the indicated times, acidified by the addition of HCl and fractionated by reversed-phase HPLC as described in Materials and Methods. The chromatograms were recorded by monitoring UV absorbance at 229 nm, and elution volumes are plotted from left to right. The topmost chromatogram in each panel is of a sample withdrawn from the reaction mixture at time zero. Only the regions of the chromatograms containing BPTI and the hydrolysis products of trypsin and BPTI are shown. Intact trypsin eluted at later times not included in the figure. The peaks containing intact inhibitor are labeled “I”.

Figure 3

Hydrolysis of BPTI variants at pH 3.4 monitored by HPLC separation. Hydrolysis reactions and chromatographic separations were carried out as described in the legend to Figure 2, except that the pH of the reaction mixture was 3.4. The peaks containing intact inhibitor and the form cleaved between residues 15 and 16 are labeled as “I” and “I*”, respectively.

Figure 4

Kinetics of hydrolysis of BPTI variants at (a) pH 7.8 and (b) pH 3.4. Hydrolysis reactions were carried out and monitored by HPLC as illustrated in Figure 2 and Figure 3. The concentrations of uncleaved inhibitor were determined by integration of the chromatogram peaks and are expressed as a percentage of the concentration at time zero. The curves represent fits of the experimental data to a first-order exponential decay function. The data corresponding to the different inhibitor forms are identified directly in panel a, and the same symbols are used in panel b. The estimated rate constants for hydrolysis at pH 7.8 are listed in Table 1. The rates at pH 3.4 were: C14S, 2.0×10⁻⁵ s⁻¹; C38S, 1.0×10⁻⁵ s⁻¹; C14S/C38S, 3.2×10⁻⁶ s⁻¹; 14SH/38SH, 2.1×10⁻⁵ s⁻¹.

Figure 5

Apparent rate constants for hydrolysis of BPTI variants by trypsin versus pH. Hydrolysis reactions were carried out at the indicated pH values and the progress of the reactions were monitored by reversed-phase HPLC, as illustrated in Figure 2 and Figure 3. Rate constants for the reactions were determined by least-squares fitting to a first-order decay function as shown in Figure 4. The curves represent the function described by Equation 7 in the text, with the parameters estimated manually. For all three of the variants, the values of k₁ and k₂ were both set to 10 s⁻¹. The values used for k⁻¹ for the three mutants were: C14S, 1×10⁸ s⁻¹; C38S, 3.5×10⁸ s⁻¹; C14S/C38S, 1.5×10⁹ s⁻¹. The pK_a values used were: C14S, pK₁ = 5.7, pK₂= 6.9; C38S, pK₁ = 5.7, pK₂ = 6.9; C14S/C38S, pK₁ = 5, pK₂ = 6.5.

Figure 6

Equilibrium binding measurements for BPTI variants and bovine trypsin. A fixed concentration of trypsin (1.3 nM) was incubated with the indicated total concentrations of inhibitor at pH 7.8 and 25 °C. The concentration of free enzyme was determined using a spectrophotometric assay with a chromogenic substrate, as described in Materials and Methods. In panel a, the curves are those predicted assuming a single binding site and a dissociation constant of either 6×10⁻¹⁴ M (the value previously determined for the wild-type inhibitor (23), dashed curve) or 6×10⁻¹² M (solid curve). In panels b and c, the curves represent least squares fits of the binding function to the experimental data. The fit values of the dissociation constants were 1.7×10⁻¹⁰ M and 1.4×10⁻¹⁰ M for the C14S and C38S variants, respectively.

Figure 7

Heats of dissociation (ΔH_d) for the complex of trypsin with C14S/C38S BPTI. Enthalpy changes were measured by isothermal titration calorimetry at pH 8 in the presence of 20 mM CaCl₂ and 50 mM Tris-HCl (filled circles) or 50 mM HEPES (open circles). The lines represent least-squares fits to the experimental data, from which estimates of the heat capacity change, ΔC_p,d, and ΔH_d at 25 °C were derived. The parameters for the data obtained with Tris-HCl are listed in Table 1. For HEPES, the parameters were: ΔH_d = 10.6±0.2 kcal/mol, ΔC_p,d = 500±20 cal/deg·mol.

Figure 8

Structures of complexes of trypsin with BPTI variants containing replacements of Cys14 or Cys38, highlighting the active site region. The orientation shown corresponds approximately to that shown in Figure 1, rotated by 90° about the vertical axis, so that the view is from the inhibitor towards the enzyme active site. The enzyme site is shown in a surface representation, and the primary binding residues of the inhibitors are represented as sticks. Carbon, nitrogen, oxygen and sulfur atoms of the inhibitor are colored white, blue, red and yellow respectively. The scissile peptide bond of the inhibitor is colored black and identified by an arrow. The surface of the side-chain oxygen of the catalytic Ser residue (Ser195) of trypsin is colored red. Electron density maps corresponding to the inhibitor residues are represented as cages, contoured at the level of 1 σ. The diffraction data for the C14S/C38S mutant were consistent with a single conformation for the two altered residues, which occupied positions nearly identical to those of the Cys sulfur atoms in the wild-type structure. For the mutants with single replacements, C14S and C38S, the calculated electron density maps indicated the presence of alternative conformations for residues 14 and 38. The refined structure of the C14S variant included the alternate conformations shown in the figure, with equal occupancy in each case. In the C38S structure, the two conformations of Cys14 were represented at equal occupancy, but Ser38 was modeled with 75% occupancy of the conformation labeled A and 25% occupancy of conformation B. The alternate conformation of Cys14 in panel d is hidden. The arrow in each panel indicates the scissile peptide bond of the inhibitor, between Lys15 and Ala16. Drawn from the atomic coordinates in PDB entries 2FI3 (C14S/C38S), 2FI4 (C14S) and 2FI5 (C38S).

Figure 9

Crystallographic temperature factors for backbone atoms of trypsin and BPTI in complexes containing the C14S/C38S, C14S and C38S BPTI variants. Average B-values for the N, C_α, C and O atoms are plotted as a function of residue number. In each panel, the solid curve represents the values for the indicated mutant, and the dashed lines are the values for the complex with wild-type BPTI (PDB entry 2FTL). The vertical dashed lines identify the average B-values for residues 14 and 38 of the inhibitors and the residues of the trypsin catalytic triad.