A cofactor approach to copper-dependent catalytic antibodies.
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.
Nicholas
Kenneth M
KM
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA. kmnicholas@chemdept.chem.ou.edu
Wentworth
Paul
P
Harwig
Curtis W
CW
Wentworth
Anita D
AD
Shafton
Asher
A
Janda
Kim D
KD
D000812:Anhydrides / Q000737:chemistry*; D017151:Antibodies, Catalytic / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D003300:Copper / Q000737:chemistry*; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry*; D007093:Imidazoles / Q000737:chemistry*; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D007659:Ketones / Q000737:chemistry; D007783:Lactones / Q000737:chemistry; D015394:Molecular Structure
D000812:Anhydrides; D017151:Antibodies, Catalytic; D000911:Antibodies, Monoclonal; D007093:Imidazoles; D007140:Immunoglobulin Fab Fragments; D007659:Ketones; D007783:Lactones; D003300:Copper; C109536:antibody aldolase; D005634:Fructose-Bisphosphate Aldolase