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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 that 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 (k(cat) = 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.

SUBMITTER: Xu D

PROVIDER: S-EPMC2741171 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Quantum mechanical/molecular mechanical and density functional theory studies of a prototypical zinc peptidase (carboxypeptidase A) suggest a general acid-general base mechanism.

Xu Dingguo D Guo Hua H

Journal of the American Chemical Society 20090701 28

Carboxypeptidase A is a zinc-containing enzyme that 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 (k(cat) = 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 ...[more]

PMID: 19552427

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Project description:Microwave driven dynamic nuclear polarization (DNP) is a process in which the large polarization present in an electron spin reservoir is transferred to nuclei, thereby enhancing NMR signal intensities. In solid dielectrics there are three mechanisms that mediate this transfer--the solid effect (SE), the cross effect (CE), and thermal mixing (TM). Historically these mechanisms have been discussed theoretically using thermodynamic parameters and average spin interactions. However, the SE and the CE can also be modeled quantum mechanically with a system consisting of a small number of spins and the results provide a foundation for the calculations involving TM. In the case of the SE, a single electron-nuclear spin pair is sufficient to explain the polarization mechanism, while the CE requires participation of two electrons and a nuclear spin, and can be used to understand the improved DNP enhancements observed using biradical polarizing agents. Calculations establish the relations among the electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) frequencies and the microwave irradiation frequency that must be satisfied for polarization transfer via the SE or the CE. In particular, if δ, Δ < ω(0I), where δ and Δ are the homogeneous linewidth and inhomogeneous breadth of the EPR spectrum, respectively, we verify that the SE occurs when ω(M) = ω(0S) ± ω(0I), where ω(M), ω(0S) and ω(0I) are, respectively, the microwave, and the EPR and NMR frequencies. Alternatively, when Δ > ω(0I) > δ, the CE dominates the polarization transfer. This two-electron process is optimized when ω(0S(1))-ω(0S(2)) = ω(0I) and ω(M)~ω(0S(1)) or ω(0S(2)), where ω(0S(1)) and ω(0S(2)) are the EPR Larmor frequencies of the two electrons. Using these matching conditions, we calculate the evolution of the density operator from electron Zeeman order to nuclear Zeeman order for both the SE and the CE. The results provide insights into the influence of the microwave irradiation field, the external magnetic field, and the electron-electron and electron-nuclear interactions on DNP enhancements.

Dataset Information

Quantum mechanical/molecular mechanical and density functional theory studies of a prototypical zinc peptidase (carboxypeptidase A) suggest a general acid-general base mechanism.

Publications

Quantum mechanical/molecular mechanical and density functional theory studies of a prototypical zinc peptidase (carboxypeptidase A) suggest a general acid-general base mechanism.

Similar Datasets

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