Biomedical subjects

Constance J Jeffery

Publications and source records attributed to Constance J Jeffery.

8 recordsLinked to original sources

PubMed2005

The crystal structure of rabbit phosphoglucose isomerase complexed with D-sorbitol-6-phosphate, an analog of the open chain form of D-glucose-6-phosphate.

Phosphoglucose isomerase (PGI) catalyzes the isomerization of D-glucose-6-phosphate (G6P) and D-fructose-6-phosphate (F6P) in glycolysis and gluconeogenesis. Analysis of previously reported X-ray crystal structures of PGI without ligand, with the cyclic form of F6P, or with inhibitors that mimic the cis-enediol intermediate led to proposed mechanisms for the ring opening and isomerization steps in the multistep catalytic mechanism. To help complete our model of the overall mechanism, information is needed about the state of PGI between the ring opening and isomerization steps, in other words, a structure of the enzyme complexed with the open form of a substrate or an analog. Here, we report the crystal structure of rabbit PGI complexed with D-sorbitol-6-phosphate (S6P), an analog of the open chain form of G6P, at 2.0 A resolution. As was seen in the PGI/F6P structure, a helix containing amino acid residues 512-520 is found in the "out" position, which provides sufficient space in the active site for a substrate in its cyclic form and which is probably the location of that helix just after ring opening (or just before ring closure). However, the S6P ligand is in an extended conformation, as was seen previously with ligands that mimic the cis-enediol intermediate. The extended conformation enables the ligand to interact with Glu357, which transfers a proton during the isomerization step. The PGI/S6P structure represents the conformation of the enzyme and substrate between the ring opening (or ring closing) step and the isomerization step and helps to complete the model for PGI's catalytic mechanism.

Animals↗

PubMed2004

Inhibition of type I and type II phosphomannose isomerases by the reaction intermediate analogue 5-phospho-D-arabinonohydroxamic acid supports a catalytic role for the metal cofactor.

The phosphomannose isomerases (PMI) comprise three families of proteins: type I, type II, and type III PMIs. Members of all three families catalyze the reversible isomerization of D-mannose 6-phosphate (M6P) and D-fructose 6-phosphate (F6P) but share little or no sequence identity. Because (1) PMIs are essential for the survival of several microorganisms, including yeasts and bacteria, and (2) the PMI enzymes from several pathogens do not share significant sequence identity to the human protein, PMIs have been considered as potential therapeutic targets. Elucidation of the catalytic and regulatory mechanisms of the different types of PMIs is strongly needed for rational species-specific drug design. To date, inhibition and crystallographic studies of all PMIs are still largely unexplored. As part of our research program on aldose-ketose isomerases, we report in this paper the evaluation of two new inhibitors of type I and type II PMIs from baker's yeast and Pseudomonas aeruginosa, respectively. We found that 5-phospho-D-arabinonohydroxamic acid (5PAH), which is the most potent inhibitor of phosphoglucose isomerase (PGI), is by far the best inhibitor ever reported of both type I and type II PMI-catalyzed isomerization of M6P to F6P. 5PAH, which has an inhibition constant at least 3 orders of magnitude smaller than that of previously reported PMI inhibitors, may be the first high-energy intermediate analogue inhibitor of the enzymes. We also tested the related molecule 5-phospho-D-arabinonate (5PAA), which is a strong competitive inhibitor of PGI, and found that it does not inhibit either PMI. All together, our results are consistent with a catalytic role for the metal cofactor in PMI activity.

Bacterial Proteins↗

PubMed2004

Molecular mechanisms for multitasking: recent crystal structures of moonlighting proteins.

Recently determined X-ray crystal structures of moonlighting proteins are helping to elucidate how a protein can evolve two different functions and, in some cases, switch between its two functions in response to cellular conditions. X-ray crystal structures of the I-AniI homing endonuclease/maturase and the PutA proline dehydrogenase/transcription factor have provided evidence that these proteins utilize separate protein surfaces for their multiple functions. Also, the structure of the DegP (HtrA) protease/chaperone has revealed information about the mechanism of its chaperone activity and suggests how the protein regulates its protease activity. Comparing the structure of eta-crystallin/retinal dehydrogenase with structures of its single-function enzyme homologs provides clues to changes in the protein structure that may have improved its ability to serve as a crystallin, but at the same time may have adversely affected its catalytic activity.

Bacterial Proteins↗

PubMed2003

Moonlighting proteins: old proteins learning new tricks.

Recently, several laboratories identifying proteins involved in the complex processes of replication, transcription and tumor suppression found that the 'new' protein they discovered had another, previously identified, function. A single protein with multiple functions might seem surprising, but there are actually many cases of proteins that 'moonlight', or have more than one role in an organism. As well as adding to the number and types of proteins that are known to moonlight, these new examples add to our understanding of the potential importance of moonlighting proteins.

Animals↗

PubMed2003

Multifunctional proteins: examples of gene sharing.

Adding to the difficulty of interpreting the human genome sequence and annotating protein sequence databases is the observation that a single protein can 'moonlight' or perform multiple, apparently unrelated, functions. This review summarizes examples of moonlighting proteins in cellular activities and biochemical pathways important in cancer and other diseases. The proteins include a variety of combinations of functions and mechanisms to switch between functions. Moonlighting proteins can be beneficial to the organism, such as by coordinating cellular activities. However, moonlighting proteins can potentially make more difficult the determination of the molecular mechanisms of disease and the process of rational drug design.

Binding Sites↗

PubMed2002

Conformational changes in phosphoglucose isomerase induced by ligand binding.

Phosphoglucose isomerase (PGI; EC 5.3.1.9) is the second enzyme in glycolysis, where it catalyzes the isomerization of D-glucose-6-phosphate to D-fructose-6-phosphate. It is the same protein as autocrine motility factor, differentiation and maturation mediator, and neuroleukin. Here, we report a new X-ray crystal structure of rabbit PGI (rPGI) without ligands bound in its active site. The structure was solved at 1.8A resolution by isomorphous phasing with a previously solved X-ray crystal structure of the rPGI dimer containing 6-phosphogluconate in its active site. Comparison of the new structure to previously reported structures enables identification of conformational changes that occur during binding of substrate or inhibitor molecules. Ligand binding causes an induced fit of regions containing amino acid residues 209-215, 245-259 and 385-389. This conformational change differs from the change previously reported to occur between the ring-opening and isomerization steps, in which the helix containing residues 513-521 moves toward the bound substrate. Differences between the liganded and unliganded structures are limited to the region within and close to the active-site pocket.

Animals↗

PubMed2002

The crystal structure of rabbit phosphoglucose isomerase complexed with 5-phospho-D-arabinonohydroxamic acid.

Phosphoglucose isomerase (EC ) catalyzes the second step in glycolysis, the reversible isomerization of D-glucose 6-phosphate to D-fructose 6-phosphate. The reaction mechanism involves acid-base catalysis with proton transfer and proceeds through a cis-enediol(ate) intermediate. 5-Phospho-D-arabinonohydroxamic acid (5PAH) is a synthetic small molecule that resembles the reaction intermediate, differing only in that it has a nitrogen atom in place of C1. Hence, 5PAH is the best inhibitor of the isomerization reaction reported to date with a K(i) of 2 x 10(-7) M. Here we report the crystal structure of rabbit phosphoglucose isomerase complexed with 5PAH at 1.9 A resolution. The interaction of 5PAH with amino acid residues in the enzyme active site supports a model of the catalytic mechanism in which Glu-357 transfers a proton between C1 and C2 and Arg-272 helps stabilize the intermediate. It also suggests a mechanism for proton transfer between O1 and O2.

Animals↗

PubMedDate not supplied

Mass spectrometry and the search for moonlighting proteins.

Mass spectrometry has become one of the most important techniques in proteomics because of its use to identify the proteins found in different cell types, organelles, and multiprotein complexes. This information about protein location and binding partners can provide valuable clues to infer a protein's function. However, more and more proteins are found that "moonlight," or have more than one function, and the presence of moonlighting proteins can make more difficult the identification of protein function in those studies. This review discusses examples of moonlighting proteins and how their presence can affect the results of mass spectrometry studies that identify the locations, levels, and changes in protein expression. Although the presence of moonlighting proteins can complicate the results of those studies, mass spectrometry-derived protein-expression profiles potentially provides a very powerful method to find additional moonlighting proteins because they do not require a prior hypothesis of the protein's function.

Gene Expression Profiling↗