The critical step in porphyrin biosynthesis is the condensation of two molecules of amino-levulinic acid (ALA) into the monopyrrole, porphobilinogen. This reaction is catalyzed by porphorbilinogen synthase (PBGS). All PBGS contain at least 8 metal ions; the mammalian enzyme is a Zn(4)Zn(4) species, with two Zn(2+) per dimeric unit in a homooctameric structure. In each homodimer, one Zn is bound to what is known as the """"""""A"""""""" site, one Zn(2+) is bound at the """"""""B"""""""" site. This proposal has four specific aims focussed on determination of the structure and catalytic mechanism of this essential enzyme.
The first aim i s to determine the chemical structure of the PBGS-catalyzed reaction intermediates. Three approaches will be used. First, site-directed mutagenesis will be used to make substitutions at residues thought to play a role as a covalent catalyst, a metal ligand, or Bronsted base/acid. Second, fluorinated substrate(s) will be used that cannot undergo turnover. Third, rapid quench approaches will be tried.
The second aim i s to determine the manner by which PBGS from various species use metal ions. This will involve the preparation of a number of PBGSs from various sources, with various combinations of Zn(2+), Mg(2+), Mn(2+), followed by analysis of metal ligation by EXAFS and ESR, and function. The inhibition of these several forms of PBGS by Pb(2+) is the focus of the third aim which is to determine whether the mechanism of lead inhibition is common to all of these enzyme forms. The last aim is to complete the x-ray structural determination of PBGS.
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