THEORETICAL-STUDY OF THE ROLE OF ARGININE-127 IN THE WATER-PROMOTED MECHANISM OF PEPTIDE CLEAVAGE BY CARBOXYPEPTIDASE-A

The water-promoted mechanism of peptide cleavage by carboxypeptidase A (CPA) has been studied by means of molecular dynamics simulations and AM1 quantum mechanical calculations. A representative molecular dynam ics structure has been used to design a realistic quantum mechanical m odel involving 106 atoms, which includes for the first time the Arg-12 7 residue (simulated by a guanidinium group) among others. In turn, th e accessibility of the conformations that are required for the quantum mechanical mechanism has been assessed from molecular dynamics simula tions involving 8274 atoms. Our results show that proton transfer to G lu-270 from the water molecule attached to the Zn ion is required as a previous step to correct substrate anchoring. Arg-127 turns out to be important for initial binding of the substrate and stabilizing the na scent negative charge appearing on the carbonyl oxygen atom during for mation of the tetrahedral intermediate when the activated water attack s the scissile peptide bond. After a suitable rotation of the substrat e, the zinc ion is already able to coordinate the negative carbonylic oxygen atom, this way reinforcing the effect of Arg-127 and leading to a more stable tetrahedral intermediate. As a consequence, the proton transfer from Glu-270 to the nitrogen atom of the breaking bond become s the step associated with the energetically highest transition state of the complete process. Finally, we feel that although quantitative v alues of enthalpy barriers could be somewhat overestimated by the AM1 Hamiltonian, the qualitative picture of the CPA catalytic mechanism de scribed in this work is likely good enough and already includes the ma in key groups of the real system.