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 subsequent identification of metal-binding sites using anomalous difference Fourier analyses once the structure of the enzyme had been solved. These sites include the highly inhibitory substitution of an enzymic cofactor Zn(2+) ion by Pb(2+) ions, which represents a major contribution towards understanding the 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-crystal and a Hg co-crystal to provide the first delineation of the enzyme's quaternary structure. In this MADIR analysis, the Hg co-crystal data were treated as native data. Anomalous difference Fouriers were again used, revealing that Hg(2+) had substituted for the same Zn(2+) cofactor ion as had Pb(2+), a finding of fundamental importance for the understanding of mercury poisoning. In addition, Pt(2+) ions were found to bind at the same place in the structure. The refined structures of the Pb- and the Hg-complexed enzymes are presented at 2.5 and 3.0 A resolution, respectively.
Erskine
P T
PT
Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, England.
Duke
E M
EM
Tickle
I J
IJ
Senior
N M
NM
Warren
M J
MJ
Cooper
J B
JB
D015502:Absorptiometry, Photon / Q000379:methods; D001665:Binding Sites; D018360:Crystallography, X-Ray / Q000379:methods; D005583:Fourier Analysis; D007854:Lead / Q000378:metabolism; D008627:Mercuric Chloride / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D009942:Organometallic Compounds / Q000378:metabolism; D000623:Porphobilinogen Synthase / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D015032:Zinc / Q000378:metabolism
D008670:Metals; D009942:Organometallic Compounds; D007854:Lead; D008627:Mercuric Chloride; D000623:Porphobilinogen Synthase; D015032:Zinc; C008261:lead acetate