Quantum Mechanical/Molecular Mechanical and Density Functional Theory Studies of a Prototypical Zinc Peptidase (Carboxypeptidase A) Suggest a General Acid−General Base Mechanism

Abstract

Carboxypeptidase A is a zinc containing enzyme which cleaves the C-terminal residue in a polypeptide substrate. Despite much experimental work, there is still a significant controversy concerning its catalytic mechanism. In this study, the carboxypeptidase A catalyzed hydrolysis of the hippuryl-L-Phe molecule (kcat=17.7±0.7 s−1) is investigated using both density functional theory and a hybrid quantum mechanical/molecular mechanical approach. The enzymatic reaction was found to proceed via a promoted-water pathway with Glu270 serving as the general base and general acid. Free-energy calculations indicate that the first nucleophilic addition step is rate-limiting, with a barrier of 17.9 kcal/mol. Besides activating the zinc-bound water nucleophile, the zinc cofactor also serves as an electrophilic catalyst that stabilizes the substrate carbonyl oxygen during the formation of the tetrahedral intermediate. In the Michaelis complex, Arg127, rather than Zn(II), is responsible for the polarization of the substrate carbonyl and it also serves as the oxyanion hole. As a result, its mutation leads to a higher free-energy barrier, in agreement with experimental observations.

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

“…The PDB contains a bound inhibitor, o-(((1r)-((n-phenylmethoxycarbonyl-l-alanyl)amino)-ethyl)hydroxyphosphono)-l-benzylacetic acid (ZAAP(O)F), which served as a scaffold to build the substrate, as done in other computational works. 46,55,56,60,63 QTAIM analysis of native and mutant active sites. The electron charge density has been shown by Kohn to be nearsighted, 64a,b meaning that r(r) can be accurately reproduced using small cluster models in the active sites of enzymes, as long as the nuclear coordinates were obtained using full protein modeling, such as the QM/DMD methods used here.…”

“…2C and E), with hydrogen bonds' placements and lengths in the substrate binding pockets that agree well with previous experimental and computational studies on the native CPA (Table 1). 46,47 In particular, the distance between the carbonyl oxygen and zinc, Zn-O1, (Fig. 1B) , producing the Zn-O1 distance of 4.19 Å.…”

“…Previous works emphasize that the exact mechanism of CPA is not fully understood, it is particularly unclear as to whether the peptide hydrolysis is accomplished through a promoted water or anhydride mechanism. However, computations [44][45][46] have shown that the water-promoted mechanism has the lowest barrier for the rate-determining step, nucleophilic attack of peptide backbone carbonyl carbon by zinc-bound water (Fig. 1A).…”

“…46 Furthermore, the residue specificity comes from the hydrophobic pocket, also known as the S' subsite, that is responsible for the preference for aromatic and aliphatic side chains. 46 Additionally, Arg127 interacts not only with the terminal carboxylic acid, but also with the carbonyl oxygen of the peptide backbone coordinated to zinc, an interaction believed to contribute to catalysis and substrate binding. 46,61 During the course of the reaction, Arg127 stabilizes the oxyanion developed on the tetrahedral intermediate.…”

“…46 Additionally, Arg127 interacts not only with the terminal carboxylic acid, but also with the carbonyl oxygen of the peptide backbone coordinated to zinc, an interaction believed to contribute to catalysis and substrate binding. 46,61 During the course of the reaction, Arg127 stabilizes the oxyanion developed on the tetrahedral intermediate. Thus, in the design, the goal is to preserve these valuable interactions.…”