Theoretical study of the role of arginine 127 in the water-promoted mechanism of peptide cleavage by carboxypeptidase A

Abstract

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 dynamics structure has been used to design a realistic quantum mechanical model involving 106 atoms, which includes for the first time the Arg-127 residue (simulated by a guanidinium group) among others. In turn, the accessibility of the conformations that are required for the quantum mechanical mechanism has been assessed from molecular dynamics simulations involving 8274 atoms. Our results show that proton transfer to Glu-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 nascent negative charge appearing on the carbonyl oxygen atom during formation of the tetrahedral intermediate when the activated water attacks the scissile peptide bond. After a suitable rotation of the substrate, 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 becomes the step associated with the energetically highest transition state of the complete process. Finally, we feel that although quantitative values of enthalpy barriers could be somewhat overestimated by the AM1 Hamiltonian, the qualitative picture of the CPA catalytic mechanism described in this work is likely good enough and already includes the main key groups of the real system.