Human porphobilinogen synthase (PBGS) is a main target in lead poisoning. H
uman PBGS purifies with eight Zn(II) per homo-octamer; four ZnA have predom
inantly nonsulfur ligands, and four ZnB have predominantly sulfur ligands.
Only four Zn(II) are required for activity. To better elucidate the roles o
f Zn(TI) and Pb(II), we produced human PBGS mutants that are designed to la
ck 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 importa
nt catalytic role for ZnB and the removed cysteine residues. Kinetic proper
ties of the mutants and wild type proteins are described, Comparison of Pb(
IT) inhibition of the mutants shows that ligands to both ZnA and ZnB intera
ct with Pb(II). The ZnB ligands preferentially interact with Pb(II), At lea
st 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 hy
brid of the ZnA and ZnB sites. We propose that the lone electron pair of Pb
(IT) precludes Pb(II) to function in PBGS catalysis.