Eileen K. Jaffe, PhD
Senior Member & Professor
Office Phone: 215 728-3695
Lab Phone: 215 728-5268
Fax: 215 728-2412
The Porphobilinogen Synthase Family
Porphobilinogen synthase (PBGS) is an ancient enzyme that catalyzes the first common step in the biosynthesis of all tetrapyrrole pigments, such as heme, chlorophyll, and vitamin B12. Our initial interest in PBGS arose from its role as a primary target for the environmental toxin lead. Human PBGS is a zinc metalloprotein whose active site zinc is coordinated to three cysteine residues, which is a rather atypical situation for zinc ions at enzyme active sites, and which forms a suburb environment for binding divalent lead.
Consequently, environmental exposure to lead causes blood PBGS to become inactivated through the substitution of lead for the active site zinc; this phenomenon has been used as a diagnostic for lead poisoning. In severe cases lead inhibition of PBGS results in anemia. The buildup of the PBGS substrate, which is a structural analog of a common neurotransmitter, results in the psychiatric disorders characteristic of lead poisoning.
More recently work from our laboratory and many others has established that the active site zinc used in human PBGS is not phylogenetically conserved. Species in the kingdom Archaea use the active site zinc, but many species in the kingdom Bacteria do not and usage among the kingdom Eukaryota is also mixed.
This variation in active site metal ion usage suggests that all PBGS may not utilize a common catalytic mechanism. One focus of the Jaffe lab is determination of the mechanistic differences between the PBGS that use the active site zinc and those that do not. In addition to a phylogenetic variation in active site metal ion usage, the PBGS family of enzymes also contains a phylogenetic variation in the use of an allosteric magnesium ion, which binds outside the active site. Although this allosteric ion is present in the PBGS of most organisms, it is notably absent in metazoa (e.g. humans), fungi (yeast), and a small number of bacteria and protozoa. It may be possible to capitalize on the phylogenetic differences in the use of metal ions by PBGS as a gateway for species specific inhibition of tetrapyrrole biosynthesis, which could serve as the basis for antimicrobials or herbicides.Top
Porphobilinogen Synthase as a Prototype Morpheein
Many proteins function as assemblies that contain multiple copies of a single subunit structure; these are homo-oligomers. It is generally taught that proteins fold and assemble into one physiologically relevant oligomer and that variations in function are associated with conformational changes that occur within the context of that oligomer. Quite unexpectedly, the Jaffe laboratory has determined that regulation of PBGS function is associated with alternate non-additive assembly states. PBGS exists as an equilibrium ensemble containing high activity octamers, low activity hexamers, and a dimeric assembly whose two different conformations dictate assembly into octamer or hexamer.
This equilibrium responds to environmental variables such as pH, which can vary with cellular localization, and to point mutations
that affect the thermodynamic stability of one or another oligomer. For many species of PBGS this equilibrium is modulated by the presence or absence of the allosteric magnesium, described above. The functional distinction between the octamer (on-state) and the hexamer (off-state) and variations in the rates of the dissociation, conformational change, and reassociation lead to kinetic behaviors such as hysteresis. The Jaffe lab has coined the word “morpheein” to describe homo-oligomeric proteins that can come apart, change conformation in the dissociated state, and reassemble to a structurally and functionally distinct oligomer. This allosteric mechanism is different from the two classic models of allostery provided by Monod (concerted model) and Koshland (sequential model).
The morpheein model for allostery requires that a homo-oligomeric protein assembly dissociate and that a conformational change occur in the dissociated state. Detailed investigation of PBGS from various species is being used to define the physical and kinetic behaviors associated with this fascinating and unexpected quaternary structure dynamic.Top
Trapping Alternate PBGS Assemblies as a Structural Basis for Novel Antimicrobial Therapies
The low activity hexameric assembly of PBGS contains a surface cavity, which s not present in the octamer. Small molecule binding to this cryptic allosteric site was predicted to stabilize the hexameric assembly, shift the equilibrium toward the low activity hexamer, and thus inhibit protein function. Because the proposed hexamer-specific allosteric site on PBGS is phylogenetically variable, it is possible to target this site for the discovery of antibiotics or herbicides. We have used a combination of in silico and in vitro techniques to discover several hexamer-stabilizing inhibitors of PBGS. In one notable example, the inhibitor is specific for a plant PBGS, does not inhibit human PBGS and has an IC50 on the order of micromolar. Small molecules (e.g. drugs) that can affect the function of a morpheein by binding to an allosteric site that is specific for one oligomeric assembly, stabilize that assembly, and thus shift the quaternary structure equilibrium, provide a novel mode of drug action. There may be many proteins that function as morpheeins for which similar drug discovery efforts can be applied.Top
The Identification of Proteins that Function as Morpheeins
Control of protein function is an essential aspect of all cellular activities. Commonly accepted methods for functional control include post-translational modifications such as phosphorylation. The existence of alternate non-additive assembly states in the absence of covalent modification, which can be accomplished by proteins that we describe as morpheeins, provides a largely untapped resource for the design or discovery of new therapeutic and/or diagnostic agents. The quaternary structure dynamic available to proteins that function as morpheeins is illustrated in the most general sense using a morphing dice analogy.
Although PBGS is the only protein for which hard physical data unequivocally establish an equilibrium of morpheein forms, it is possible, if not probable, that other proteins use a similar morpheein mechanism for allosteric regulation. Small molecule stabilization of one or another assembly state is proposed as a general approach to shifting such an equilibrium, as we have done for PBGS. One ongoing project in the Jaffe laboratory is the identification of other proteins that might function as morpheeins, with an emphasis on those whose pharmaceutical modulation addresses unmet medical need. As there is no predictive relationship between quaternary structure dynamics and protein sequence information, efficient identification of proteins that function as morpheeins from the literature and protein structure databases requires development of novel techniques in text and data mining.Top