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

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

“…It plays an important role in metabolic pathways by catalysing the cleavage of C terminal peptide bonds, favouring those which have large bulky hydrophobic side chains, such as phenylalanine and tyrosine. CPA is a typical zinc hydrolase and thus many studies, both experimental [1,2] and theoretical [3,4] have been carried out to understand its structure and mechanism. Developing a comprehensive knowledge of CPA is potentially beneficial in terms of its applications to other zinc hydrolase enzymes.…”

Predictive methods for computational metalloenzyme redesign – a test case with carboxypeptidase A

“…40 Several computational works, based on small cluster models and sometimes semi-empirical methods, have shed light on the mechanism of peptide hydrolysis in CPA. [41][42][43][44] The water-promoted mechanism, schematically depicted in Fig. 1A, is believed to be the most energetically favorable.…”

“…1A, is believed to be the most energetically favorable. 43,44 An alternative mechanism proposed for CPA, called the anhydride mechanism, is thought to compete with the water-promoted mechanism for hydrolysis of non-natural substrates, such as esters.…”