MAD analyses of yeast 5-aminolaevulinate dehydratase: their use in structure determination and in defining the metal-binding sites

MAD experiments attempting to solve the structure of 5-aminolaevulinic acid dehydratase using Zn and Pb edges are described. The data obtained proved insufficient for a complete structure solution but were invaluable in subse quent identification of metal-binding sites using anomalous difference Four ier analyses once the structure of the enzyme had been solved. These sites include the highly inhibitory substitution of an enzymic cofactor Zn2+ ion by Pb2+ ions, which represents a major contribution towards understanding t he molecular basis of lead poisoning. The MAD data collected at the Pb edge were also used with isomorphous replacement data from the same Pb co-cryst al and a Hg cocrystal to provide the first delineation of the enzyme's quat ernary structure. In this MADIR analysis, the Hg cocrystal data were treate d as native data. Anomalous difference Fouriers were again used, revealing that Hg2+ had substituted for the same Zn2+ cofactor ion as had Pb2+, a fin ding of fundamental importance for the understanding of mercury poisoning. In addition, Pt2+ ions were found to bind at the same place in the structur e. The refined structures of the P-band the Hg-complexed enzymes are presen ted at 2.5 and 3.0 Angstrom resolution, respectively.