A COMBINATORIAL LIBRARY FOR THE BINUCLEAR METAL CENTER OF BACTERIAL PHOSPHOTRIESTERASE

Phosphotriesterase (PTE) is a zinc metalloenzyme that catalyzes the hy drolysis of an extensive array of organophosphate pesticides and mamma lian acetylcholinesterase nerve agents. Although the three-dimensional crystal structure of PTE has been solved (M. M. Benning et al., Bioch emistry 34:7973-7978, 1995), the precise functions of the individual a mino acid residues that interact directly with the substrate at the ac tive site are largely unknown. To construct mutants of PTE with altere d specificities for particular target substrates, a simple methodology for generating a library of mutants at specific sites was developed. In this investigation, four of the six protein ligands to the binuclea r metal site (His-55, His-57, His-201, and His-230) were targeted for further characterization and investigation. Using the polymerase chain reaction (PCR) protocols, a library of modified PTE genes was generat ed by simultaneously creating random combinations of histidine and cys teine codons at these four positions. The 16 possible DNA sequences we re isolated and confirmed by dideoxy-DNA sequencing. The 16 mutant pro teins were expressed in Escherichia coli and grown with the presence o r absence of 1 mM CoCl2, ZnSO4, or CdSO4 in the growth medium. When gr own in the presence of CoCl2, the H57C protein cell. lysate showed gre ater activity for the hydrolysis of paraoxon than the wild type PTE ce ll lysate. H201C and H230C exhibited up to 15% of the wild-type activi ty, while H55C, a green protein, was inactive under all assay conditio ns. All other mutants had <10(-5) of wild-type activity. None of the p urified mutants that exhibited catalytic activity had a significantly altered K-m for paraoxon. (C) 1997 Wiley-Liss, Inc.