Porphobilinogen synthase (PBGS) is an ancient protein, essential to nearly all-cellular organisms. PBGS catalyzes the first common step in tetrapyrrole biosynthesis (i.e., porphyrin, chlorophyll, vitamin B12), which is the condensation of two molecules of 5-aminolevulinic acid to form porphobilinogen. Despite multiple crystal structures, the order and identity of the catalytic steps remains unclear. The PBGS family consists of metalloproteins among which the utilization of metal ions has a unique phylogenetic variation between Zn2+, Mg2+, and K+. Three hypotheses drive the proposed studies. First, we propose that there are mechanistic differences between the PBGS that use Zn2+ and those that do not. Second, we propose that there is a physiological significance to our newly observed hexameric form of PBGS. All previously deposited crystal structures show an octamer. Third, we propose that a subunit-to-subunit communication outside the active site is the structural basis for the half-site reactivity evident in PBGS. Four interrelated Aims address these hypotheses. Aim 1 is directed at elucidating the human Zn2+-PBGS catalyzed reaction mechanism. One novel approach uses a variant designed to process two different substrates, instead of two ALA molecules. We propose to use kinetic techniques, isotope effect determinations, 13C and 15N NMR, and other collaborative approaches. Aim 2 focuses on the newly discovered hexameric form of PBGS. A hexamer-octamer transition is proposed to be the structural basis for allosteric activation by Mg 2+ for some PBGS. We will also probe for a relationship between altemate quaternary forms of the protein and an allelic variation reported to predispose some humans toward the environmental disease of lead poisoning. Aim 3 describes investigation of the Mg2+ requiring PBGS where we focus on mechanism, structure, and the quaternary structure equilibria. Aim 4 focuses on Drosophila melanogaster PBGS, which is a superior system for the study of the Zn 2+ utilizing PBGS and an excellent model for probing the subunit-to-subunit communication required for half-site reactivity. In support of these aims are collaborations to probe the structure and function of PBGS using X-ray crystallography, Raman spectroscopy, and analytical ultracentrifugation.