ISOLATION AND CRYSTALLIZATION OF FUNCTIONALLY COMPETENT ESCHERICHIA-COLI PEPTIDE DEFORMYLASE FORMS CONTAINING EITHER IRON OR NICKEL IN THE ACTIVE-SITE

Three metallo forms of peptide deformylase (PDF, EC 3.5.1.31) of Esche richia colt were prepared and crystallized (space group C2, diffractio n limit 1.9 Angstrom) for initiating the X-ray structure determination of the metal center in correlation with the catalytic function ality of this enzyme. The native Fe2+ containing enzyme species was directly isolated from overproducing bacteria by using catalase as a buffer ad ditive, which stabilizes the catalytic activity against oxidative dest ruction. The Ni2+ containing form, which is oxygen-insensitive, was ob tained by metal exchange with free Ni+2 and found to be catalytically equally effective (k(cat)/K-M = 10(5) M-1 s(-1) for N-formyl-Met-Ala). The Zn2+ form, prepared from the apoenzyme or by displacement of boun d Ni2+ by free Zn2+, proved virtually inactive. (C) 1998 Academic Pres s.