The molecular mechanism of lead inhibition of human porphobilinogen synthase.
Human porphobilinogen synthase (PBGS) is a main target in lead poisoning. Human PBGS purifies with eight Zn(II) per homo-octamer; four ZnA have predominantly nonsulfur ligands, and four ZnB have predominantly sulfur ligands. Only four Zn(II) are required for activity. To better elucidate the roles of Zn(II) and Pb(II), we produced human PBGS mutants that are designed to lack either the ZnA or ZnB sites. These proteins, MinusZnA (H131A, C223A) and MinusZnB (C122A, C124A, C132A), each become purified with four Zn(II) per octamer, thus confirming an asymmetry in the human PBGS structure. MinusZnA is fully active, whereas MinusZnB is far less active, verifying an important catalytic role for ZnB and the removed cysteine residues. Kinetic properties of the mutants and wild type proteins are described. Comparison of Pb(II) inhibition of the mutants shows that ligands to both ZnA and ZnB interact with Pb(II). The ZnB ligands preferentially interact with Pb(II). At least one ZnA ligand is responsible for the slow tight binding behavior of Pb(II). The data support a novel model where a high affinity lead site is a hybrid of the ZnA and ZnB sites. We propose that the lone electron pair of Pb(II) precludes Pb(II) to function in PBGS catalysis.
Jaffe
E K
EK
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA. EK_Jaffe@fccc.edu
Martins
J
J
Li
J
J
Kervinen
J
J
Dunbrack
R L
RL
D019943:Amino Acid Substitution; D001665:Binding Sites; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007854:Lead / Q000494:pharmacology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D000623:Porphobilinogen Synthase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D017384:Sequence Deletion; D015032:Zinc / Q000378:metabolism*
D011994:Recombinant Proteins; D007854:Lead; D000623:Porphobilinogen Synthase; D015032:Zinc