On the water-promoted mechanism of peptide cleavage by carboxypeptidase A. A theoretical study

Abstract

SILVIA ALVAREZ-SANTOS, ANGELS GONZ~FZ-LAFONT, JOSB M. LLUCH, BALDOMERO OLNA, and FRANCE~C X. A m & . Can. J. Chem. 72,2077Chem. 72, (1994.The water-promoted pathway of peptide cleavage by carboxypeptidase A has been studied by semiempirical (AM1) quantum mechanical calculations. A relative1 large model for the CPA-active site elements plus substrate has been designed, using two 3+ .imidazoles and one acetate as the Zn Iigands, acetate as the proton acceptor (simulating , and N-ethylacetamide as the peptide-like substrate. This model, although simpler than the natural one, is one of the largest used for theoretical calculations on CPA catalytic mechanisms. To ensure that this model is able to mimic the natural system, it has been compared with the structure of the ( G I Y )~-L -T~~ + water + CPA complex resulting from several molecular dynamicslenergy minimization simulations.Among the different steps involved in the water-promoted pathway proposed by Lipscomb's group, the attack of the oxygen atom that comes from the activated water molecule to the carbon atom of the peptide bond of the substrate has been found to be the rate-determining step, with a high enthalpy bamer of 37.9 kcal/mol. However, this enthalpy bamer is dramatically decreased when a positive charge, simulating Arg-127, is included near the scissile carbonyl. The reported results seem to favour the occurrence of the mechanism studied and indicate the limitations of using simple elements for the theoretical analysis of enzyme-catalyzed reactions.SUVIA ALVAREZ-SANTOS, ANGELS GONZ~EZ-LAFONT, JOSB M. LLUCH, BALDOMERO OLNA et FRANCE~C X. Avu&. Can. J. Chem. 72,2077 (1994).Utilisant des calculs semiempiriques de micanique quantique (AMl), on a ttudit la voie de clivage des peptides par la carboxypeptidase A (CPA), catalysCe par l'eau. On a mis au point un modble relativement grand des ClCments du site actif de la CPA avec le substrat qui utilise deux imidazoles et un acitate comme ligands du zn2+, un acCtate comme accepteur de proton (simulant le Glu-270) et le N-CthylacCtamide comme substrat semblable i un peptide. Ce modble, mbme s'il est plus simple que le modble naturel, est un des plus grand qui a Ct C utilisC pour des calculs thCoriques sur les mCcanismes catalytiques de la CPA. Afin de s'assurer que ce modble peut imiter le comportement du systbme naturel, on l'a compare avec la structure du complexe ( G~Y )~-L-Tyr + eau + CPA qui rtsulte de plusieurs simulations de dynamique molCculaire et minimisation de l'energie. Parmi les diffkrentes Ctapes impliqutes dans la voie catalysCe par l'eau proposCe par le groupe de Lipscomb, on a trouvC que 1'Ctape determinant la vitesse de la rkaction est l'attaque de l'atome d'oxygbne, provenant de la molCcule d'eau activCe, sur l'atome de carbone de la liaison peptidique du substrat et qu'elle est accompagnCe d'une bambre enthalpique Clevte (37,9 kcal/mol). Toutefois, il se produit une diminution dramatique de cette barribre lorsqu'on introduit une charge positive, ressemblant i celle de

Assessment of a mechanism for reactive inhibition of carboxypeptidase A with QM/MM methods

“…The same QM/MM protocol, described earlier for the inhibitor studies, was followed using the AM1 hamiltonian; in this case a QM active site which not only included the imidazole groups of His-69 and His-196, and the carboxylate side chains of Glu-72 and Glu-270, but also the catalytic water, and the full GLT substrate. A transition state corresponding to nucleophilic attack at the carbonyl of GLT by hydroxyl, with the water proton mostly dissociated onto Glu-270, was found and compared with the corresponding step in the AM1 study by Alvarez-Santos; in our case the activation barrier of 33 kcal mol À1 is in fair agreement with the previously published barriers, 28 kcal mol À1 [8] and 24 kcal mol À1 [9], considering that the QM region used here was smaller than the QM gas phase models used in the previous studies.…”

“…Although competing mechanisms for CPA catalysis with various substrates have been proposed from both experimental and computational studies [8][9][10][11] it is now widely accepted that CPA cleaves the C-terminal amino acid residue of a natural peptide substrate, e.g. glycyl-L-tyrosine (GLT), via a general acid-base mechanism involving an activated water molecule.…”

QM/MM study on the mechanism of peptide hydrolysis by carboxypeptidase A

“…Their initial study yielded a reaction barrier of 37.9 kcal/mol [11], however a subsequent study which included a guanidinium group to represent Arg127 yielded a significantly lower barrier of 19.9 kcal/mol [3]. This difference was accounted for by consideration of the stabilisation effect the arginine residue has on the negatively charged oxygen atom upon formation of the tetrahedral intermediate.…”

On the origin of the catalytic power of carboxypeptidase A and other metalloenzymes

“…Alex and Clark 52 constructed a simplified model of CPA and performed AM1 calculations for the different steps suggested for the water-promoted pathway proposed by Lipscomb’s group 13. Alvarez-Santos et al 53 designed a model of the active site of CPA plus substrate, containing 51 atoms and identified the water (hydroxide) attack on peptide carbonyl as the rate-determining step, with a high enthalpy barrier of 37.9 kcal/mol. This calculated activation enthalpy was dramatically decreased when a positive charge (that represented Arg127) was included.…”