The catalytic aspartate is protonated in the Michaelis complex formed between trypsin and an in vitro evolved substrate-like inhibitor: a refined mechanism of serine protease action

Abstract

The mechanism of serine proteases prominently illustrates how charged amino acid residues and proton transfer events facilitate enzyme catalysis. Here we present an ultrahigh resolution (0.93 Å) x-ray structure of a complex formed between trypsin and a canonical inhibitor acting through a substrate-like mechanism. The electron density indicates the protonation state of all catalytic residues where the catalytic histidine is, as expected, in its neutral state prior to the acylation step by the catalytic serine. The carboxyl group of the catalytic aspartate displays an asymmetric electron density so that the O(δ2)-C(γ) bond appears to be a double bond, with O(δ2) involved in a hydrogen bond to His-57 and Ser-214. Only when Asp-102 is protonated on O(δ1) atom could a density functional theory simulation reproduce the observed electron density. The presence of a putative hydrogen atom is also confirmed by a residual mF(obs) - DF(calc) density above 2.5 σ next to O(δ1). As a possible functional role for the neutral aspartate in the active site, we propose that in the substrate-bound form, the neutral aspartate residue helps to keep the pK(a) of the histidine sufficiently low, in the active neutral form. When the histidine receives a proton during the catalytic cycle, the aspartate becomes simultaneously negatively charged, providing additional stabilization for the protonated histidine and indirectly to the tetrahedral intermediate. This novel proposal unifies the seemingly conflicting experimental observations, which were previously seen as either supporting the charge relay mechanism or the neutral pK(a) histidine theory.

FIGURE 1.

Stereo diagram showing SGPI-1-PO-2 bound to the surface of bovine cationic trypsin colored by the electrostatic potential (red negative, blue positive). a, the Glu-I5, Arg-I18, Gly-I20, Ser-I21, Asp-I22, Arg-I30, and Met-I31 residues of the inhibitor are highlighted. Basic residues (Arg-I18 and Arg-I30) and acidic residue Asp-I22 are marked as blue and red, respectively, whereas neutral residues Gly-I20, Ser-I21, and Met-I31 are colored orange. For other amino acid residues of the inhibitor, only the backbone is displayed (yellow). It is evident that the mutations N18R, T22D, and K31M improve the complementarity to the surface charges of bovine cationic trypsin. b, SGPI-1-PO-2 (yellow) and SGTI (gray) in the enzyme-bound form (43) are superimposed and reveal a similar fold. The mutation sites are highlighted with the same colors as in a.

FIGURE 2.

Comparison of the theoretical protonated and deprotonated carboxyl groups to experimental electron density in the crystal structure at 0.93-Å resolution. a, electron density of Asp-102 compared with theoretical protonated propionic acid. In the DFT calculation, the H-O-C-C torsional angle was constrained to 109° to match the putative hydrogen position observed in the mF_obs − DF_calc electron density. b, electron density of Asp-189 compared with theoretical deprotonated propionic acid. Theoretical electron densities of propionic acid are contoured at 2.35 e⁻/ų, whereas the experimental 2mF_obs − DF_calc electron density map (blue) is contoured at 3.01 e⁻/ų (4.5 σ), and the positive mF_obs − DF_calc density map (green) is contoured at 0.22 e⁻/ų (2.5 σ).

FIGURE 3.

Alternative protonation states of the imidazole ring as calculated by density functional theory and experimentally observed at residue His-57 in the electron density maps at 0.93-Å resolution. The DFT electron density of 4-ethyl-imidazole is contoured at 2.01 e⁻/ų, whereas the 2mF_obs − DF_calc (blue) and mF_obs − DF_calc (green) electron density maps at 2.34 e⁻/ų (3.5 σ) and 0.26 e⁻/ų (3.0 σ), respectively. The experimentally observed electron density is more similar to an unprotonated imidazole ring.

FIGURE 4.

Schematic representation of the proposed catalytic mechanism. Left, Michaelis complex with both His-57 and Asp-102 in the neutral state. Right, the tetrahedral intermediate state when both His-57 and Asp-102 becomes charged.

FIGURE 5.

a, ESCET structural comparison of SGTI and SGPI-1-PO-2. Blue in the matrix marks C_α atoms that are closer to one another in the SGPI-1-PO-2 structure than in SGTI (44), whereas red represents C_α atom pairs that are further away from one another. Apart from the C and N termini, two extended regions (residues ranges I26-I32 and I20-I25) appear to be closer to one another in SGPI-1-PO-2. b, these regions are illustrated in a stereo diagram of the superpositioned SGTI and SGPI-1-PO-2 inhibitors. The coloring scheme follows Fig. 1 with SGPI-1-PO-2 (yellow) and SGTI (gray), but in addition regions I20-I25 and I26-I32 in SGPI-1-PO-2 are highlighted as magenta and green, respectively.