A cofactor approach to copper-dependent catalytic antibodies

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 CuCl 2, or (ii) precoordination of 5 with CuCl 2 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 [k cat(11)kuncat (11) C atalysis at metal centers plays a key role in both enzymatic and abiological reactions, providing reaction pathways, rates, and selectivity often unattainable from conventional acid, base, or nucleophilic catalysis. However, traditionally separate disciplines, the study of natural (i.e., enzymatic) and synthetic (nonbiological) catalysts has interfaced with the genesis of semisynthetic and de novo proteins (1). A number of methods have been explored for producing such hybrid species that possess metal centers, including: (i) attachment of a synthetic metal-containing cofactor to a protein (2-4); (ii) antibody elicitation using a metal-binding hapten͞protein conjugate (5-10); (iii) site-directed mutagenesis of proteins and antibodies to incorporate metal-binding sites (11-15); and (iv) panning of antibody libraries for metal binding using immobilized metal complexes (16,17).Our approach is directed toward the design and generation of novel metallo-catalytic antibodies and is inspired by the active-site structures of the many metalloenzymes that possess a coordination sphere with two or more histidine-derived imidazole ligands. The most important catalytic functions of such polyhistidyl enzymes are hydrolytic or oxidative. Illustrative of the numerous and structurally diverse polyhistidyl Zn-hydrolases (18) is carboxypeptidase A, which catalyzes the hydrolysis of C-terminal amino acid residues and features a bis-histidine͞glutamate (CO 2 Ϫ ) coordination sphere and the nucleophilic assistance of a nearby Glu (19). The Cuhydroxylases, dopamine ␤-hydroxylase and peptide amidating hydroxylase (20), are chemically extraordinary in effecting the regio-and enantioselective hydroxylation of weakly activated COH bonds, a transformation with little precedent among nonenzymatic copper catalysts (21,22). The active sites of hydroxylase enzymes possess two Cu-polyhistidine centers, one of which appears to serve as an electron or superoxide transfer site and the other at which the substra...

Molecular characterization of the copper transport system in Staphylococcus aureus

“…Copper is an essential trace element required by most organisms as a cofactor for numerous catabolic pathways and electron transport (Mason, 1976;Nicholas et al, 2002;Massaro, 2002). However, copper is toxic to cells at concentrations higher than physiological levels (Gaetke & Chow, 2003).…”

Interfacial metal and antibody recognition

“…Abs can distinguish conformational alterations induced by metals in antigens and have been designed or selected to bind metals by direct ligation (11) or indirectly through metal-binding cofactors (12)(13)(14)(15). Many of the latter cases of ''designed'' metal binders have involved ''catalytic'' Abs (16).…”

Artificial metalloenzymes as selective catalysts in aqueous media

“…3e). An attractive method to obtain natively evolved proteins designed for distinct transition metal complexes is the preparation of antibodies against the chosen catalyst [39,[100][101][102][103]. This area has been reviewed elsewhere [97] and thus only one recent example is presented.…”