A cofactor approach to copper-dependent catalytic antibodies.
Proc Natl Acad Sci U S A
; 99(5): 2648-53, 2002 Mar 05.
Article
em En
| MEDLINE
| ID: mdl-11880619
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fragmentos Fab das Imunoglobulinas
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Anticorpos Catalíticos
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Cobre
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Frutose-Bifosfato Aldolase
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Imidazóis
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Anidridos
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Anticorpos Monoclonais
Idioma:
En
Ano de publicação:
2002
Tipo de documento:
Article