Computational investigation of irreversible inactivation of the zinc-dependent protease carboxypeptidase A

Jason B. Cross, Thom Vreven, Samy Meroueh, Shahriar Mobashery, H. Bernhard Schlegel

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Zinc proteases are ubiquitous and the zinc ion plays a central function in the catalytic mechanism of these enzymes. A novel class of mechanism-based inhibitors takes advantage of the zinc ion chemistry in carboxypeptidase A (CPA) to promote covalent attachment of an inhibitor to the carboxylate of Glu-270, resulting in irreversible inhibition of the enzyme. The effect of the active site zinc ion on irreversible inactivation of CPA was probed by molecular orbital (MO) calculations on a series of active site models and the Cl - + CH 3Cl S N2 reaction fragment. Point charge models representing the active site reproduced energetics from full MO calculations at 12.0 Å separation between the zinc and the central carbon of the S N2 reaction, but at 5.0 Å polarization played an important role in moderating barrier suppression. ONIOM MO/MO calculations that included the residues within 10 Å of the active site zinc suggest that about 75% of the barrier suppression arises from the zinc ion and its ligands. A model of the pre-reactive complex of the 2-benzyl-3-iodopropanoate inactivator with CPA was constructed from the X-ray structure of L-phenyl lactate bound in the active site of the enzyme. The model was fully solvated and minimized by using the AMBER force field to generate the starting structure for the ONIOM QM/MM calculations. Optimization of this structure led to the barrierless S N2 displacement of the iodide of the inhibitor by Glu-270, assisted by interaction of the zinc ion with the leaving group. The resulting product is in good agreement with the X-ray structure of the covalently modified enzyme obtained by irreversible inhibition of CPA by 2-benzyl-3-iodopropanoate.

Original languageEnglish (US)
Pages (from-to)4761-4769
Number of pages9
JournalJournal of Physical Chemistry B
Volume109
Issue number10
DOIs
StatePublished - Mar 17 2005
Externally publishedYes

Fingerprint

Carboxypeptidases A
protease
deactivation
Zinc
Peptide Hydrolases
zinc
Molecular orbitals
Orbital calculations
Ions
enzymes
molecular orbitals
Enzymes
inhibitors
Enzyme inhibition
ions
retarding
X rays
lactates
Iodides
iodides

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry

Cite this

Computational investigation of irreversible inactivation of the zinc-dependent protease carboxypeptidase A. / Cross, Jason B.; Vreven, Thom; Meroueh, Samy; Mobashery, Shahriar; Schlegel, H. Bernhard.

In: Journal of Physical Chemistry B, Vol. 109, No. 10, 17.03.2005, p. 4761-4769.

Research output: Contribution to journalArticle

Cross, Jason B. ; Vreven, Thom ; Meroueh, Samy ; Mobashery, Shahriar ; Schlegel, H. Bernhard. / Computational investigation of irreversible inactivation of the zinc-dependent protease carboxypeptidase A. In: Journal of Physical Chemistry B. 2005 ; Vol. 109, No. 10. pp. 4761-4769.
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