A catalysis-based selection for peroxidase antibodies with increased activity.

A biotin-tyramine conjugate (1) was found to covalently cross-link with peroxidase antibody 7G12 upon the catalytic oxidation of the tyramine moiety in the presence of hydrogen peroxide (H2O2). On the basis of this observation, a novel strategy was developed to select mutants of 7G12 Fab with enhanced peroxidase activity from a library of phage displayed antibodies. In such a selection, tyramine is oxidized by hydrogen peroxide in a process catalyzed by peroxidase antibodies displayed on phage. Antibodies with higher peroxidase activity are preferentially labeled with biotin through irreversible adduct formation between oxidized biotin-linked tyramine molecules and phenolic side chains of the antibody. The corresponding phage particles can then be selected via biotin-streptavidin interactions. Using this strategy, phage displayed libraries of antibody 7G12 were selected for higher peroxidase activity. As a result, mutations of antibody 7G12 that led to 10 to 20-fold increases in the peroxidase activity (kcat/Km) were identified, suggesting the validity of this method for the evolution of peroxidase antibodies based directly on catalytic turnover.

Structural and kinetic evidence for strain in biological catalysis.

A classic hypothesis for enzyme catalysis is the induction of strain in the substrate. This notion was first expressed by Haldane with the lock and key analogy-"the key does not fit the lock perfectly but exercises a certain strain on it" (1). This mechanism has often been invoked to explain the catalytic efficiency of enzymes but has been difficult to establish conclusively (2-7). Here we describe X-ray crystallographic and mutational studies of an antibody metal chelatase which strongly support the notion that this antibody catalyzes metal ion insertion into the porphyrin ring by inducing strain. Analysis of the germline precursor suggests that this strain mechanism arose during the process of affinity maturation in response to a conformationally distorted N-alkylmesoporphyrin.

Alternative modes of substrate distortion in enzyme and antibody catalyzed ferrochelation reactions.

Both an antibody that catalyzes metal insertion into porphyrins and the corresponding enzyme, ferrochelatase, are shown by resonance Raman spectroscopy to induce distortion in the bound porphyrin substrate. It was found that the enzyme-induced distortion is different from that induced by the antibody; the catalytic antibody produces a distortion which is similar to the one present in the hapten, N-methylmesoporphyrin IX (N-MeMP). Activation of specific out-of-plane vibrational modes reveal that the antibody induces an alternating up-and-down tilting of the pyrrole rings, while ferrochelatase induces tilting of all four pyrrole rings in the same direction (doming). Both distortions are effective in catalyzing metal insertion. The distortion induced in the enzyme is only seen when an inhibitory metal ion is also bound. This observation suggests an allosteric mechanism, in which a conformational change which distorts the porphyrin toward the transition state geometry, is induced by metal binding at an adjacent site. In contrast, the antibody does not have a metal binding site and appears to function largely through binding interactions with the porphyrin.

Antibody-catalyzed porphyrin metallation.

An antibody elicited to a distorted N-methyl porphyrin catalyzed metal ion chelation by the planar porphyrin. At fixed Zn2+ and Cu2+ concentrations, the antibody-catalyzed reaction showed saturation kinetics with respect to the substrate mesoporphyrin IX (2) and was inhibited by the hapten, N-methylmesoporphyrin IX (1). The turnover number of 80 hour-1 for antibody-catalyzed metallation of 2 with Zn2+ compares with an estimated value of 800 hour-1 for ferrochelatase. The antibody also catalyzed the insertion of Co2+ and Mn2+ into 2, but it did not catalyze the metallation of protoporphyrin IX (3) or deuteroporphyrin IX (4). The antibody has high affinity for several metalloporphyrins, suggesting an approach to developing antibody-heme catalysts for redox or electron transfer reactions.