Biomedical subjects

Eileen K Jaffe

Publications and source records attributed to Eileen K Jaffe.

12 recordsLinked to original sources

PubMed2005

Single amino acid mutations alter the distribution of human porphobilinogen synthase quaternary structure isoforms (morpheeins).

Porphobilinogen synthase (PBGS) is an obligate oligomer that can exist in functionally distinct quaternary states of different stoichiometries, which are called morpheeins. The morpheein concept describes an ensemble of quaternary structure isoforms wherein different structures of the monomer dictate different multiplicities of the oligomer (Jaffe, E. K. (2005) Trends Biochem. Sci. 30, 490-497). Human PBGS assembles into long-lived morpheeins and has been shown to be capable of forming either a high activity octamer or a low activity hexamer (Breinig, S., Kervinen, J., Stith, L., Wasson, A. S., Fairman, R., Wlodawer, A., Zdanov, A., and Jaffe, E. K. (2003) Nat. Struct. Biol. 10, 757-763). All PBGS monomers contain an alphabeta-barrel domain and an N-terminal arm domain. The N-terminal arm structure varies among PBGS morpheeins, and the spatial relationship between the arm and the barrel dictates the different quaternary assemblies. We have analyzed the structures of human PBGS morpheeins for key interactions that would be predicted to affect the oligomeric assembly. Examples of individual mutations that shift assembly of human PBGS away from the native octamer are R240A and W19A. The alternate morpheeins of human PBGS variants R240A and W19A are chromatographically separable from each other and kinetically distinct; their structure and dynamics have been characterized by native gel electrophoresis, dynamic light scattering, and analytical ultracentrifugation. R240A assembles into a metastable hexamer, which can undergo a reversible conversion to the octamer in the presence of substrate. The metastable nature of the R240A hexamer supports the hypothesis that octameric and hexameric morpheeins of PBGS are very close in energy. W19A assembles into a mixture of dimers, which appear to be stable.

Amino Acid Substitution↗

PubMed2005

delta-Aminolevulinate dehydratase (ALAD) porphyria: the first case in North America with two novel ALAD mutations.

The molecular basis of the enzymatic defect responsible for delta-aminolevulinate dehydratase (ALAD) porphyria (ADP) was investigated in a 14-year-old male who presented clinical and laboratory findings typical of ADP. Nucleotide sequence analysis of ALAD cDNAs from the proband revealed two novel mutations, a 265G to A base transition (C1) and a 394C to T base transition (C2), resulting in amino acid substitutions, Glu89Lys and Cys132Arg, respectively. Both mutations were present within exon 5 of the ALAD gene, and appeared to influence the binding of zinc to the enzyme which is essential for enzyme activity. It was found that the C1 mutation was inherited from his father, while the C2 mutation was from his mother. Expression of these mutant ALAD cDNAs in Chinese hamster ovary cells produced normal ALAD mRNA levels, but markedly decreased ALAD protein and enzyme activity. These results suggest that the combination of the two aberrant ALADs with little enzyme activity accounts for the markedly decreased ALAD activity observed in the proband. This case represents the molecular analysis of the ALAD gene defects in the first case of ADP identified in North America, who is a compound heterozygote for two novel ALAD gene defects.

Adolescent↗

PubMed2005

Substrate-induced interconversion of protein quaternary structure isoforms.

Human porphobilinogen synthase (PBGS) can exist in two dramatically different quaternary structure isoforms, which have been proposed to be in dynamic equilibrium. The quaternary structure isoforms of PBGS result from two alternative conformations of the monomer; one monomer structure assembles into a high activity octamer, whereas the other monomer structure assembles into a low activity hexamer. The kinetic behavior of these oligomers led to the hypothesis that turnover facilitates the interconversion of the oligomeric structures. The current work demonstrates that the interactions of ligands at the enzyme active site promote the structural interconversion between human PBGS quaternary structure isoforms, favoring formation of the octamer. This observation illustrates that the assembly and disassembly of oligomeric proteins can be facilitated by the protein motions that accompany enzymatic catalysis.

Humans↗

PubMed2005

Morpheeins--a new structural paradigm for allosteric regulation.

Classic models for the allosteric regulation of protein function consider an equilibrium among protein structures of constant oligomeric multiplicity. The morpheein (mor-phee'-in) concept expands this model to include a dynamic equilibrium of protein structures wherein a protein monomer can exist in more than one conformation and each monomer conformation dictates a different quaternary structure of finite multiplicity and different functionality. The morpheein concept provides a new framework for understanding allosteric regulation, kinetic cooperativity and hysteresis. Porphobilinogen synthase constitutes a prototype morpheein ensemble comprising several interconverting quaternary structure isoforms; one monomer conformation dictates assembly of a high-activity octamer, whereas an alternative monomer conformation dictates assembly of a low-activity hexamer. It is proposed here that the behavior of some other allosteric enzymes reflect dynamic morpheein equilibrium systems and six candidate proteins are enumerated.

Allosteric Regulation↗

PubMed2005

Abrogation of fibroblast activation protein enzymatic activity attenuates tumor growth.

Tumor-associated fibroblasts are functionally and phenotypically distinct from normal fibroblasts that are not in the tumor microenvironment. Fibroblast activation protein is a 95 kDa cell surface glycoprotein expressed by tumor stromal fibroblasts, and has been shown to have dipeptidyl peptidase and collagenase activity. Site-directed mutagenesis at the catalytic site of fibroblast activation protein, Ser624 --> Ala624, resulted in an approximately 100,000-fold loss of fibroblast activation protein dipeptidyl peptidase (DPP) activity. HEK293 cells transfected with wild-type fibroblast activation protein, enzymatic mutant (S624A) fibroblast activation protein, or vector alone, were inoculated subcutaneously into immunodeficient mouse to assess the contribution of fibroblast activation protein enzymatic activity to tumor growth. Overexpression of wild-type fibroblast activation protein showed growth potentiation and enhanced tumorigenicity compared with both fibroblast activation protein S624A and vector-transfected HEK293 xenografts. HEK293 cells transfected with fibroblast activation protein S624A showed tumor growth rates and tumorigenicity potential similar only to vector-transfected HEK293. In vivo assessment of fibroblast activation protein DPP activity of these tumors showed enhanced enzymatic activity of wild-type fibroblast activation protein, with only baseline levels of fibroblast activation protein DPP activity in either fibroblast activation protein S624A or vector-only xenografts. These results indicate that the enzymatic activity of fibroblast activation protein is necessary for fibroblast activation protein-driven tumor growth in the HEK293 xenograft model system. This establishes the proof-of-principle that the enzymatic activity of fibroblast activation protein plays an important role in the promotion of tumor growth, and provides an attractive target for therapeutics designed to alter fibroblast activation protein-induced tumor growth by targeting its enzymatic activity.

Alanine↗

PubMed2004

Rhodobacter capsulatus porphobilinogen synthase, a high activity metal ion independent hexamer.

BACKGROUND: The enzyme porphobilinogen synthase (PBGS), which is central to the biosynthesis of heme, chlorophyll and cobalamins, has long been known to use a variety of metal ions and has recently been shown able to exist in two very different quaternary forms that are related to metal ion usage. This paper reports new information on the metal ion independence and quaternary structure of PBGS from the photosynthetic bacterium Rhodobacter capsulatus. RESULTS: The gene for R. capsulatus PBGS was amplified from genomic DNA and sequencing revealed errors in the sequence database. R. capsulatus PBGS was heterologously expressed in E. coli and purified to homogeneity. Analysis of an unusual phylogenetic variation in metal ion usage by PBGS enzymes predicts that R. capsulatus PBGS does not utilize metal ions such as Zn2+, or Mg2+, which have been shown to act in other PBGS at either catalytic or allosteric sites. Studies with these ions and chelators confirm the predictions. A broad pH optimum was determined to be independent of monovalent cations, approximately 8.5, and the Km value shows an acidic pKa of approximately 6. Because the metal ions of other PBGS affect the quaternary structure, gel permeation chromatography and analytical ultracentrifugation experiments were performed to examine the quaternary structure of metal ion independent R. capsulatus PBGS. The enzyme was found to be predominantly hexameric, in contrast with most other PBGS, which are octameric. A protein concentration dependence to the specific activity suggests that the hexameric R. capsulatus PBGS is very active and can dissociate to smaller, less active, species. A homology model of hexameric R. capsulatus PBGS is presented and discussed. CONCLUSION: The evidence presented in this paper supports the unusual position of the R. capsulatus PBGS as not requiring any metal ions for function. Unlike other wild-type PBGS, the R. capsulatus protein is a hexamer with an unusually high specific activity when compared to other octameric PBGS proteins.

Amino Acid Sequence↗

PubMed2004

The porphobilinogen synthase catalyzed reaction mechanism.

Porphobilinogen synthase (PBGS) catalyzes the first common reaction in the biosynthesis of the tetrapyrroles, the asymmetric condensation of two molecules of delta-aminolevulinic acid to form porphobilinogen. There is a variable requirement for an essential active site zinc that necessitates consideration of PBGS as an enzyme that may exhibit phylogenetic diversity in its chemical reaction mechanism. Recent crystal structures suggest reaction mechanisms that involve two covalent Schiff base linkages between adjacent active site lysine residues and each of the two substrate molecules. The reaction appears to stall at a covalently bound almost-product intermediate that is poised for breakdown to product upon binding of a substrate molecule to an adjacent active site and a subsequent conformational change.

Catalysis↗

PubMed2003

Control of tetrapyrrole biosynthesis by alternate quaternary forms of porphobilinogen synthase.

Porphobilinogen synthase (PBGS) catalyzes the first common step in the biosynthesis of tetrapyrroles (such as heme and chlorophyll). Although the predominant oligomeric form of this enzyme, as inferred from many crystal structures, is that of a homo-octamer, a rare human PBGS allele, F12L, reveals the presence of a hexameric form. Rearrangement of an N-terminal arm is responsible for this oligomeric switch, which results in profound changes in kinetic behavior. The structural transition between octamer and hexamer must proceed through an unparalleled equilibrium containing two different dimer structures. The allosteric magnesium, present in most PBGS, has a binding site in the octamer but not in the hexamer. The unprecedented structural rearrangement reported here relates to the allosteric regulation of PBGS and suggests that alternative PBGS oligomers may function in a magnesium-dependent regulation of tetrapyrrole biosynthesis in plants and some bacteria.

Allosteric Site↗

PubMed2003

A structural basis for half-of-the-sites metal binding revealed in Drosophila melanogaster porphobilinogen synthase.

Porphobilinogen synthase (PBGS) proteins fall into several distinct groups with different metal ion requirements. Drosophila melanogaster porphobilinogen synthase (DmPBGS) is the first non-mammalian metazoan PBGS to be characterized. The sequence shows the determinants for two zinc binding sites known to be present in both mammalian and yeast PBGS, proteins that differ in the exhibition of half-of-the-sites metal binding. The pH-dependent activity of DmPBGS is uniquely affected by zinc. A tight binding catalytic zinc binds at 0.5/subunit with a Kd well below microm. A second inhibitory zinc exhibits a Kd of approximately 5 microm and appears to bind at a stoichiometry of 1/subunit. A molecular model of DmPBGS suggests that the inhibitory zinc is located at a subunit interface using Cys-219 and His-10 as ligands. Zinc binding to this previously unknown inhibitory site is proposed to inhibit opening of the active site lid. As predicted, the DmPBGS mutant H10F is active but is not inhibited by zinc. H10F binds a catalytic zinc at 0.5/subunit and binds a second nonessential and noninhibitory zinc at 0.5/subunit. This result reveals a structural basis for half-of-the-sites metal binding that is consistent with a reciprocating motion model for function of oligomeric PBGS.

Animals↗

PubMed2003

An unusual phylogenetic variation in the metal ion binding sites of porphobilinogen synthase.

Porphobilinogen synthase (PBGS), which catalyzes the first common step in tetrapyrrole biosynthesis, contains a unique phylogenetic variation in the use of metal ions. Using sequence, structure, and enzymological information, this work codifies the phylogenetic segregation of metal utilization in PBGS from archaea, bacteria, and eucarya. All PBGS contain an active site metal binding sequence, determined herein to be either DXCXCX(Y/F)X(3)G(H/Q)CG or DXALDX(Y/F)X(3)G(H/Q)DG. The former dictates a requirement for zinc. Most PBGS that do not require zinc require magnesium and/or potassium instead. Most PBGS are also found to contain the binding determinants for an allosteric magnesium that resides outside the active site. The phylogenetic distribution of PBGS metal ion utilization suggests that the primordial PBGS required zinc and supports a hypothesis that the loss of the zinc site was concurrent with the advent of oxygenic photosynthesis.

Allosteric Site↗

PubMed2002

Investigations on the metal switch region of human porphobilinogen synthase.

Porphobilinogen synthase (PBGS) is an ancient and highly conserved protein that functions in the first common step in tetrapyrrole biosynthesis. The PBGS protein sequence contains a unique metal switch region that has been postulated to dictate an exclusive catalytic use of either zinc or magnesium, and perhaps also potassium. In some PBGS, the cysteines of the metal switch sequence DXCXCX(Y/F)X(3)G(H/Q)CG have been demonstrated to bind a catalytic zinc, and in other PBGS, the aspartic acid residues of the metal switch sequence DXALDX(Y/F)X(3)G(H/Q)DG have been postulated to bind a catalytically essential magnesium and/or potassium. The current work describes chimeric proteins that contain the aspartate-rich sequences of pea PBGS and Pseudomonas aeruginosa PBGS in place of the naturally occurring cysteine-rich sequence of human PBGS. The resultant chimeric PBGS proteins, peainhuman PBGS and psuinhuman PBGS, are substantially activated by both magnesium and potassium, but not by zinc. The specific activities of the chimeras are significantly lower than human PBGS. Detailed kinetic and inhibition data are presented for both chimeric proteins and are discussed in terms of this unique phylogenetic variation in metal ion usage. The identity of a basic residue, which is Arg221 in human PBGS, strictly correlates with the presence or absence of the cysteine-rich sequence. Those PBGS with the aspartate-rich metal switch sequence contain Lys in the analogous position. The R221K mutation was inserted into wild type and chimeric human PBGS and found to further reduce the activity of both, illustrating the subtle nature of the role of this residue.

Amino Acid Sequence↗

PubMed2002

Species-specific inhibition of porphobilinogen synthase by 4-oxosebacic acid.

Porphobilinogen synthase (PBGS) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA), an essential step in tetrapyrrole biosynthesis. 4-Oxosebacic acid (4-OSA) and 4,7-dioxosebacic acid (4,7-DOSA) are bisubstrate reaction intermediate analogs for PBGS. We show that 4-OSA is an active site-directed irreversible inhibitor for Escherichia coli PBGS, whereas human, pea, Pseudomonas aeruginosa, and Bradyrhizobium japonicum PBGS are insensitive to inhibition by 4-OSA. Some variants of human PBGS (engineered to resemble E. coli PBGS) have increased sensitivity to inactivation by 4-OSA, suggesting a structural basis for the specificity. The specificity of 4-OSA as a PBGS inhibitor is significantly narrower than that of 4,7-DOSA. Comparison of the crystal structures for E. coli PBGS inactivated by 4-OSA versus 4,7-DOSA shows significant variation in the half of the inhibitor that mimics the second substrate molecule (A-side ALA). Compensatory changes occur in the structure of the active site lid, which suggests that similar changes normally occur to accommodate numerous hybridization changes that must occur at C3 of A-side ALA during the PBGS-catalyzed reaction. A comparison of these with other PBGS structures identifies highly conserved active site water molecules, which are isolated from bulk solvent and implicated as proton acceptors in the PBGS-catalyzed reaction.

Amino Acid Sequence↗