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A cofactor approach to copper-dependent catalytic antibodies

Academic Article
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Overview

authors

  • Nicholas, K. M.
  • Wentworth Jr., Paul
  • Harwig, C. W.
  • Wentworth, A. D.
  • Shafton, A.
  • Janda, Kim

publication date

  • March 2002

journal

  • Proceedings of the National Academy of Sciences of the United States of America  Journal

abstract

  • A strategy for the preparation of semisynthetic copper(II)-based catalytic metalloproteins is described in which a metal-binding bis-imidazole cofactor is incorporated into the combining site of the aldolase antibody 38C2. Antibody 38C2 features a large hydrophobic-combining site pocket with a highly nucleophilic lysine residue, Lys(H93), that can be covalently modified. A comparison of several lactone and anhydride reagents shows that the latter are the most effective and general derivatizing agents for the 38C2 Lys residue. A bis-imidazole anhydride (5) was efficiently prepared from N-methyl imidazole. The 38C2-5-Cu conjugate was prepared by either (i) initial derivatization of 38C2 with 5 followed by metallation with CuCl2, or (ii) precoordination of 5 with CuCl2 followed by conjugation with 38C2. The resulting 38C2-5-Cu conjugate was an active catalyst for the hydrolysis of the coordinating picolinate ester 11, following Michaelis-Menten kinetics [kcat(11) = 2.3 min(-1) and Km(11) 2.2 mM] with a rate enhancement [kcat(11)k(uncat)(11)] of 2.1 x 10(5). Comparison of the second-order rate constants of the modified 38C2 and the Cu(II)-bis-imidazolyl complex k(6-CuCl2) gives a rate enhancement of 3.5 x 10(4) in favor of the antibody complex with an effective molarity of 76.7 M, revealing a significant catalytic benefit to the binding of the bis-imidazolyl ligand into 38C2.

subject areas

  • Anhydrides
  • Antibodies, Catalytic
  • Antibodies, Monoclonal
  • Copper
  • Fructose-Bisphosphate Aldolase
  • Imidazoles
  • Immunoglobulin Fab Fragments
  • Ketones
  • Lactones
  • Molecular Structure
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Identity

PubMed Central ID

  • PMC122402

International Standard Serial Number (ISSN)

  • 0027-8424

Digital Object Identifier (DOI)

  • 10.1073/pnas.052001099

PubMed ID

  • 11880619
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Additional Document Info

start page

  • 2648

end page

  • 2653

volume

  • 99

issue

  • 5

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