The active site of papain. All-atom study of interactions with protein matrix and solvent.

The proton transfer between the Cys25 and His159 residues in the active centre of the proteolytic enzyme papain is investigated with the Hartree-Fock SCF direct reaction field method. The active centre is treated quantum mechanically, while the environment is represented by interacting partial charges and polarizabilities. All protein atoms around the active site are included explicitly in the calculations. In this way a complete description is given of both the electrostatic and the dielectric properties of the enzyme. The protein matrix stabilizes the zwitterionic form of Cys-His, which is thought to be the catalytically active state much more than the neutral configuration. The most important contribution to the stabilization comes from the alpha-helix to which Cys25 is attached; more than half of its effect is due to the backbone atoms of Cys25 itself. Other important factors are the Asn175 side-chain and the solvent. Solvent effects are estimated by means of Monte Carlo calculations of crystal water molecules that are located near the active site. The total energies of the neutral and zwitterionic structures are similar, confirming the idea that a zwitterion can exist in the active centre of papain. The energy difference, however, is sensitive to the geometry of the active site, suggesting that the two structures are in thermal equilibrium. Classical analogues of the quantum mechanical interaction energy, employing point charge representations of the active site, are found to be quite useful. The dielectric behaviour of the protein is much more complicated than is implicated in dielectric constant models; force fields that do not include an atomic level representation of electronic polarization are inadequate.