Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 12 de 12
Filter
Add more filters










Publication year range
1.
Medchemcomm ; 4(9)2013 Sep.
Article in English | MEDLINE | ID: mdl-24324854

ABSTRACT

The bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) from the protozoal parasite Cryptosporidium hominis is a potential molecular target for the design of antiparasitic therapies for AIDS-related opportunistic infections. The enzyme exists as a homodimer with each monomer containing a unique swap domain known as a "crossover helix" that binds in a cleft on the adjacent DHFR active site. This crossover helix is absent in species containing monofunctional forms of DHFR such as human. An in-depth understanding of protein-protein interactions between the crossover helix and adjacent DHFR active site that might modulate enzyme integrity or function would allow for insights into rational design of species-specific allosteric inhibitors. Mutational analysis coupled with structural studies and biophysical and kinetic characterization of crossover helix mutants identifies this domain as essential for full enzyme stability and catalytic activity, and pinpoints these effects to distinct faces of the crossover helix important in protein-protein interactions. Moreover, targeting this helical protein interaction with α-helix mimetics of the crossover helix leads to selective inhibition and destabilization of the C. hominis TS-DHFR enzyme, thus validating this region as a new avenue to explore for species-specific inhibitor design.

2.
Biochemistry ; 52(41): 7305-7317, 2013 Oct 15.
Article in English | MEDLINE | ID: mdl-24053355

ABSTRACT

Most species, such as humans, have monofunctional forms of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) that are key folate metabolism enzymes making critical folate components required for DNA synthesis. In contrast, several parasitic protozoa, including Toxoplasma gondii , contain a unique bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) having the catalytic activities contained on a single polypeptide chain. The prevalence of T. gondii infections across the world, especially for those immunocompromised, underscores the need to understand TS-DHFR enzyme function and to find new avenues to exploit for the design of novel antiparasitic drugs. As a first step, we have solved the first three-dimensional structures of T. gondii TS-DHFR at 3.7 Å and of a loop truncated TS-DHFR, removing several flexible surface loops in the DHFR domain, improving resolution to 2.2 Å. Distinct structural features of the TS-DHFR homodimer include a junctional region containing a kinked crossover helix between the DHFR domains of the two adjacent monomers, a long linker connecting the TS and DHFR domains, and a DHFR domain that is positively charged. The roles of these unique structural features were probed by site-directed mutagenesis coupled with presteady state and steady state kinetics. Mutational analysis of the crossover helix region combined with kinetic characterization established the importance of this region not only in DHFR catalysis but also in modulating the distal TS activity, suggesting a role for TS-DHFR interdomain interactions. Additional kinetic studies revealed that substrate channeling occurs in which dihydrofolate is directly transferred from the TS to DHFR active site without entering bulk solution. The crystal structure suggests that the positively charged DHFR domain governs this electrostatically mediated movement of dihydrofolate, preventing release from the enzyme. Taken together, these structural and kinetic studies reveal unique, functional regions on the T. gondii TS-DHFR enzyme that may be targeted for inhibition, thus paving the way for designing species specific inhibitors.


Subject(s)
Multienzyme Complexes/chemistry , Protozoan Proteins/chemistry , Tetrahydrofolate Dehydrogenase/chemistry , Thymidylate Synthase/chemistry , Toxoplasma/enzymology , Binding Sites , Catalysis , Catalytic Domain , Folic Acid/chemistry , Folic Acid/metabolism , Imaging, Three-Dimensional , Kinetics , Models, Molecular , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Protein Structure, Secondary , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Substrate Specificity , Tetrahydrofolate Dehydrogenase/genetics , Tetrahydrofolate Dehydrogenase/metabolism , Thymidylate Synthase/genetics , Thymidylate Synthase/metabolism , Toxoplasma/chemistry , Toxoplasma/genetics
3.
Blood ; 113(19): 4656-66, 2009 May 07.
Article in English | MEDLINE | ID: mdl-19190247

ABSTRACT

Therapeutic options for advanced B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) are limited. Available treatments can also deplete T lymphocytes, leaving patients at risk of life-threatening infections. In the National Cancer Institute cell line screen, the structurally unique natural product silvestrol produces an unusual pattern of cytotoxicity that suggests activity in leukemia and selectivity for B cells. We investigated silvestrol efficacy using primary human B-leukemia cells, established B-leukemia cell lines, and animal models. In CLL cells, silvestrol LC(50) (concentration lethal to 50%) is 6.9 nM at 72 hours. At this concentration, there is no difference in sensitivity of cells from patients with or without the del(17p13.1) abnormality. In isolated cells and whole blood, silvestrol is more cytotoxic toward B cells than T cells. Silvestrol causes early reduction in Mcl-1 expression due to translational inhibition with subsequent mitochondrial damage, as evidenced by reactive oxygen species generation and membrane depolarization. In vivo, silvestrol causes significant B-cell reduction in Emu-Tcl-1 transgenic mice and significantly extends survival of 697 xenograft severe combined immunodeficient (SCID) mice without discernible toxicity. These data indicate silvestrol has efficacy against B cells in vitro and in vivo and identify translational inhibition as a potential therapeutic target in B-cell leukemias.


Subject(s)
B-Lymphocytes/drug effects , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Triterpenes/pharmacology , Animals , Apoptosis/drug effects , B-Lymphocytes/metabolism , Blotting, Western , Chromosome Deletion , Chromosomes, Human, Pair 17/genetics , Female , Humans , In Vitro Techniques , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Male , Mice , Mice, Inbred C3H , Mice, SCID , Mice, Transgenic , Mitochondria/metabolism , Mitochondria/pathology , Myeloid Cell Leukemia Sequence 1 Protein , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Protein Biosynthesis/drug effects , Proto-Oncogene Proteins/physiology , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Survival Rate , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Transplantation, Heterologous
4.
Biochem J ; 417(3): 757-64, 2009 Feb 01.
Article in English | MEDLINE | ID: mdl-18851711

ABSTRACT

In contrast with most species, including humans, which have monofunctional forms of the folate biosynthetic enzymes TS (thymidylate synthase) and DHFR (dihydrofolate reductase), several pathogenic protozoal parasites, including Cryptosporidium hominis, contain a bifunctional form of the enzymes on a single polypeptide chain having both catalytic activities. The crystal structure of the bifunctional enzyme TS-DHFR C. hominis reveals a dimer with a 'crossover helix', a swap domain between DHFR domains, unique in that this helical region from one monomer makes extensive contacts with the DHFR active site of the other monomer. In the present study, we used site-directed mutagenesis to probe the role of this crossover helix in DHFR catalysis. Mutations were made to the crossover helix: an 'alanine-face' enzyme in which the residues on the face of the helix close to the DHFR active site of the other subunit were mutated to alanine, a 'glycine-face' enzyme in which the same residues were mutated to glycine, and an 'all-alanine' helix in which all residues of the helix were mutated to alanine. These mutant enzymes were studied using a rapid transient kinetic approach. The mutations caused a dramatic decrease in the DHFR activity. The DHFR catalytic activity of the alanine-face mutant enzyme was 30 s(-1), the glycine-face mutant enzyme was 17 s(-1), and the all-alanine helix enzyme was 16 s(-1), all substantially impaired from the wild-type DHFR activity of 152 s(-1). It is clear that loss of helix interactions results in a marked decrease in DHFR activity, supporting a role for this swap domain in DHFR catalysis. The crossover helix provides a unique structural feature of C. hominis bifunctional TS-DHFR that could be exploited as a target for species-specific non-active site inhibitors.


Subject(s)
Cryptosporidium/enzymology , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , Tetrahydrofolate Dehydrogenase/chemistry , Tetrahydrofolate Dehydrogenase/metabolism , Thymidylate Synthase/chemistry , Thymidylate Synthase/metabolism , Animals , Catalysis , Catalytic Domain , Crystallography, X-Ray , Kinetics , Models, Molecular , Multienzyme Complexes/genetics , Mutagenesis, Site-Directed , Mutation , Protein Conformation , Protozoan Proteins/genetics , Structure-Activity Relationship , Tetrahydrofolate Dehydrogenase/genetics , Thymidylate Synthase/genetics
5.
Bioorg Med Chem Lett ; 19(2): 418-23, 2009 Jan 15.
Article in English | MEDLINE | ID: mdl-19059777

ABSTRACT

The essential enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) is a validated drug target for many pathogens, but has been elusive in Cryptosporidium hominis, as active site inhibitors of the enzymes from related parasitic protozoa show decreased potency and lack of species specificity over the human enzymes. As a rational approach to discover novel inhibitors, we conducted a virtual screen of a non-active site pocket in the DHFR linker region. From this screen, we have identified and characterized a noncompetitive inhibitor, flavin mononucleotide (FMN), with micromolar potency that is selective for ChTS-DHFR versus the human enzymes. These results describe a novel allosteric pocket amenable to inhibitor targeting, and a lead compound with which to move towards potent, selective inhibitors of ChTS-DHFR.


Subject(s)
Cryptosporidium/enzymology , Enzyme Inhibitors/pharmacology , Folic Acid Antagonists/pharmacology , Tetrahydrofolate Dehydrogenase/drug effects , Allosteric Site , Animals , Catalytic Domain , Models, Molecular , Tetrahydrofolate Dehydrogenase/chemistry
6.
Biochemistry ; 47(34): 8902-11, 2008 Aug 26.
Article in English | MEDLINE | ID: mdl-18672899

ABSTRACT

The essential enzyme TS-DHFR from Cryptosporidium hominis undergoes an unusually rapid rate of catalysis at the conserved TS domain, facilitated by two nonconserved residues, Ala287 and Ser290, in the folate tail-binding region. Mutation of these two residues to their conserved counterparts drastically affects multiple steps of the TS catalytic cycle. We have determined the crystal structures of all three mutants (A287F, S290G, and A287F/S290G) in complex with active site ligands dUMP and CB3717. The structural data show two effects of the mutations: an increased distance between the ligands in the active site and increased flexibility of the folate ligand in the partially open enzyme state that precedes conformational change to the active catalytic state. The latter effect is able to be rescued by the mutants containing the A287F mutation. In addition, the conserved water network of TS is altered in each of the mutants. The structural results point to a role of the folate tail-binding residues in closely positioning ChTS ligands and restricting ligand flexibility in the partially open state to allow for a rapid transition to the active closed state and enhanced rate of catalysis. These results provide an explanation on how folate tail-binding residues at one end of the active site affect long-range interactions throughout the TS active site and validate these residues as targets for species-specific drug design.


Subject(s)
Cryptosporidium/enzymology , Multienzyme Complexes/metabolism , Protozoan Proteins/metabolism , Tetrahydrofolate Dehydrogenase/metabolism , Thymidylate Synthase/metabolism , Animals , Binding Sites/genetics , Catalysis , Cryptosporidium/genetics , Crystallography, X-Ray , Multienzyme Complexes/chemistry , Multienzyme Complexes/genetics , Mutation , Protein Binding , Protein Structure, Tertiary , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Tetrahydrofolate Dehydrogenase/chemistry , Tetrahydrofolate Dehydrogenase/genetics , Thymidylate Synthase/chemistry , Thymidylate Synthase/genetics
7.
J Immunol ; 179(10): 7121-7, 2007 Nov 15.
Article in English | MEDLINE | ID: mdl-17982104

ABSTRACT

LPS stimulates monocytes/macrophages through the activation of signaling events that modulate the production of inflammatory cytokines. Apigenin, a flavonoid abundantly found in fruits and vegetables, exhibits anti-proliferative and anti-inflammatory activities through poorly defined mechanisms. In this study, we demonstrate that apigenin inhibits the production of proinflammatory cytokines IL-1beta, IL-8, and TNF in LPS-stimulated human monocytes and mouse macrophages. The inhibitory effect on proinflammatory cytokine production persists even when apigenin is administered after LPS stimulation. Transient transfection experiments using NF-kappaB reporter constructs indicated that apigenin inhibits the transcriptional activity of NF-kappaB in LPS-stimulated mouse macrophages. The classical proteasome-dependent degradation of the NF-kappaB inhibitor IkappaBalpha was observed in apigenin LPS-stimulated human monocytes. Using EMSA, we found that apigenin does not alter NF-kappaB-DNA binding activity in human monocytes. Instead we show that apigenin, as part of a non-canonical pathway, regulates NF-kappaB activity through hypophosphorylation of Ser536 in the p65 subunit and the inactivation of the IKK complex stimulated by LPS. The decreased phosphorylation on Ser536 observed in LPS-stimulated mouse macrophages treated with apigenin was overcome by the over-expression of IKKbeta. In addition, our studies indicate that apigenin inhibits in vivo LPS-induced TNF and the mortality induced by lethal doses of LPS. Collectively, these findings suggest a molecular mechanism by which apigenin suppresses inflammation and modulates the immune response in vivo.


Subject(s)
Apigenin/pharmacology , Cytokines/immunology , Lipopolysaccharides/toxicity , Macrophages/immunology , Monocytes/immunology , Protein Processing, Post-Translational/drug effects , Transcription Factor RelA/immunology , Animals , Cell Line , Cytokines/biosynthesis , Humans , I-kappa B Kinase/immunology , I-kappa B Kinase/metabolism , I-kappa B Proteins/immunology , I-kappa B Proteins/metabolism , Inflammation/chemically induced , Inflammation/immunology , Inflammation/metabolism , Macrophages/metabolism , Macrophages/pathology , Male , Mice , Monocytes/metabolism , NF-KappaB Inhibitor alpha , Phosphorylation/drug effects , Proteasome Endopeptidase Complex/immunology , Proteasome Endopeptidase Complex/metabolism , Protein Processing, Post-Translational/immunology , Transcription Factor RelA/metabolism , Transcription, Genetic/drug effects , Transcription, Genetic/immunology
8.
Biochemistry ; 46(28): 8379-91, 2007 Jul 17.
Article in English | MEDLINE | ID: mdl-17580969

ABSTRACT

Cryptosporidium hominis TS-DHFR exhibits an unusually high rate of catalysis at the TS domain, at least 10-fold greater than those of other TS enzymes. Using site-directed mutagenesis, we have mutated residues Ala287 and Ser290 in the folate-binding helix to phenylalanine and glycine, respectively, the corresponding residues in human and most other TS enzymes. Our results show that the mutant A287F, the mutant S290G, and the double mutant all have reduced affinities for methylene tetrahydrofolate and reduced rates of reaction at the TS domain. Interestingly, the S290G mutant enzyme had the lowest TS activity, with a catalytic efficiency approximately 200-fold lower than that of the wild type (WT). The rate of conformational change of the S290G mutant is approximately 80 times slower than that of WT, resulting in a change in the rate-limiting step from hydride transfer to covalent ternary complex formation. We have determined the crystal structure of ligand-bound S290G mutant enzyme, which shows that the primary effect of the mutation is an increase in the distance between the TS ligands. The kinetic and crystal structure data presented here provide the first evidence explaining the unusually fast TS rate in C. hominis.


Subject(s)
Alanine/chemistry , Cryptosporidium/enzymology , Multienzyme Complexes/chemistry , Protozoan Proteins/chemistry , Serine/chemistry , Tetrahydrofolate Dehydrogenase/chemistry , Thymidylate Synthase/chemistry , Alanine/genetics , Amino Acid Sequence , Animals , Binding Sites , Catalysis , Chromatography, High Pressure Liquid , Crystallography, X-Ray , Folic Acid/analogs & derivatives , Folic Acid/chemistry , Kinetics , Models, Molecular , Molecular Sequence Data , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Mutagenesis, Site-Directed , Protein Structure, Tertiary , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Quinazolines/chemistry , Serine/genetics , Tetrahydrofolate Dehydrogenase/genetics , Tetrahydrofolate Dehydrogenase/metabolism , Tetrahydrofolates/chemistry , Thymidine Monophosphate/metabolism , Thymidylate Synthase/genetics , Thymidylate Synthase/metabolism
9.
Biochem Pharmacol ; 72(6): 681-92, 2006 Sep 14.
Article in English | MEDLINE | ID: mdl-16844095

ABSTRACT

Apigenin, a flavone abundantly found in fruits and vegetables, exhibits antiproliferative, anti-inflammatory, and antimetastatic activities through poorly defined mechanisms. In the present study, the treatment of different cell lines with apigenin resulted in selective antiproliferative and apoptotic effect in monocytic and lymphocytic leukemias. Apigenin-induced-apoptosis was mediated by the activation of caspase-9 and caspase-3. Apigenin was found intracellularly and localized to the mitochondria. Treatment of monocytic cells with apigenin was accompanied by an increase in reactive oxygen species (ROS) and phosphorylation of the MAPKs, p38 and ERK. However, the inhibition of ROS, p38 or ERK failed to block apoptosis, suggesting that these cellular responses induced by apigenin are not essential for the induction of apoptosis. In addition, apigenin induced the activation of PKCdelta. Pharmacological inhibition of PKCdelta, the expression of dominant-negative PKCdelta and silencing of PKCdelta in leukemia cells showed that apigenin-induced-apoptosis requires PKCdelta activity. Together, these results indicate that this flavonoid provides selective activity to promote caspase-dependent-apoptosis of leukemia cells and uncover an essential role of PKCdelta during the induction of apoptosis by apigenin.


Subject(s)
Apigenin/pharmacology , Apoptosis/drug effects , Caspases/physiology , Protein Kinase C-delta/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , 3T3 Cells , Animals , Apoptosis/physiology , Caspase 3 , Caspase 9 , Caspases/metabolism , Enzyme Activation/drug effects , HL-60 Cells , Humans , Jurkat Cells , K562 Cells , Leukemia/pathology , Mice , Mitochondria/metabolism , Reactive Oxygen Species/metabolism , Tumor Cells, Cultured , U937 Cells
10.
Biochem Pharmacol ; 71(11): 1629-36, 2006 May 28.
Article in English | MEDLINE | ID: mdl-16620786

ABSTRACT

2, 3, 5, 6-Tetrachloro-1, 4-benzoquinone (TCBQ) is a metabolite of pentachlorophenol known to react with cysteines of glutathione transferases (GSTs). TCBQ treatment of rat kidney rGSTA1-2 and rGSTA1-1 abolishes 70-80% conjugation of glutathione (GSH) to 1-chloro-2, 4-dinitrobenzene and results in strongly correlated quenching of intrinsic fluorescence of Trp-20 (R>0.96). rGSTA2-2 is only inhibited by 25%. Approximately 70% (rGSTA1-1) and 60% (rGSTA1-2) conjugation activity is abolished at TCBQ: GST stoichiometries near 1:1. The inactivation follows a Kitz/Wilson model with K(D) of 4.77+/-2.5microM for TCBQ and k(3) for inactivation of 0.036+/-0.01min(-1). A single tryptic peptide labelled with TCBQ was isolated from kidney rGSTA1-2 containing Cys-17 which we identify as the site of modification. Treatment with more than stoichiometric amounts of TCBQ modified other residues but resulted in only modest further inhibition of catalysis. We interpret these findings in terms of localised steric effects on the relatively rigid alpha-helix 1 adjacent to the catalytic site of subunit 1 possibly affecting the Alpha class-specific alpha-helix 9 which acts as a "lid" on the hydrophobic part of the active site. Homology modelling of rGSTA1-1 modified at Cys-17 of one subunit revealed only modest structural perturbations in the second subunit and tends to exclude global structural effects.


Subject(s)
Chloranil/pharmacology , Glutathione Transferase/chemistry , Glutathione/metabolism , Isoenzymes/chemistry , Kidney/enzymology , Animals , Catalytic Domain , Glutathione Transferase/antagonists & inhibitors , Glutathione Transferase/metabolism , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Ligands , Male , Models, Molecular , Protein Subunits , Rats , Rats, Wistar
11.
Protein Sci ; 13(6): 1586-93, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15152091

ABSTRACT

Heterodimers of rat glutathione S-transferase A1-1 were formed using one wild-type subunit and one subunit with a mutation at the interface to evaluate whether the subunits are interactive or independent. Within the subunit interface, we are considering two regions of interactions: one region consists of a "hydrophobic ball and socket" with Phe 52 from one subunit as the ball and Phe 136 from the second subunit as one of the socket residues. The second region of interaction consists of Arg 69 and Glu 97 from both subunits. The heterodimers were formed after incubation in 1,6-hexanediol. Because one subunit in each pair had a His-tag, the heterodimers were purified using a nickel-nitrilotriacetic acid column. The specific activities of the heterodimer were compared with those of the two homodimers to determine whether the less active, mutant subunit communicates with the other subunit. Two of the heterodimers, wild type/R69E-His and wild type/E97Q-His, displayed specific activities much lower than that expected for independent active sites; in these cases, there are new close repulsive interactions and the low activity of one subunit is communicated to the neighboring subunit. In contrast, the other two heterodimers, wild type/R69Q-His and F136A/wild type-His, exhibited specific activities similar to those expected for independent active sites; in these heterodimers, the closest interaction is not repulsive or occurs over a much longer distance and the subunits act independently. We conclude that whether the subunits interact or are independent depends on the nature of the interactions at the subunit interface.


Subject(s)
Glutathione Transferase/chemistry , Glutathione Transferase/genetics , Mutation/genetics , Animals , Binding Sites , Dimerization , Gene Expression , Glutathione Transferase/isolation & purification , Glutathione Transferase/metabolism , Isoenzymes , Models, Molecular , Protein Binding , Protein Structure, Quaternary , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/isolation & purification , Protein Subunits/metabolism , Rats , Sequence Analysis, Protein , Substrate Specificity
12.
Biochemistry ; 43(12): 3327-35, 2004 Mar 30.
Article in English | MEDLINE | ID: mdl-15035604

ABSTRACT

Alpha class glutathione S-transferase, isozyme A1-1, is a dimer (51 kDa) of identical subunits. Using the crystal structure, two main areas of subunit interaction were chosen for study: (1) the hydrophobic ball and socket comprised of Phe52 from one subunit fitting into a socket formed on the other subunit by Met94, Phe136, and Val139 and (2) the Arg/Glu region consisting of Arg69 and Glu97 from both subunits. We introduced substitutions of these residues, by site-directed mutagenesis, to evaluate the importance of each at the subunit interface and to determine if monomeric enzymes could be generated using single mutations. Mutating each residue of the socket region to alanine results in little change in the kinetic parameters, and all are dimeric enzymes. In contrast, when Phe52, the ball residue, is replaced with alanine, the enzyme has very low activity and a weight average molecular mass of 31.9 kDa, as determined by sedimentation equilibrium experiments. Substitutions for Glu97 which eliminate the charge cause no appreciable changes in the kinetic parameters or molecular mass. Eliminating the charge on Arg69 (as in R69Q) results in a dimeric enzyme; however, when the charge is reversed (as in R69E), the weight average molecular mass is greatly shifted toward that of the monomer (33 kDa) and the changes in kinetic parameters are reasonably small. We determined the molecular masses in the presence of glutathione for F52A and R69E to ascertain whether the monomeric species retains activity. For R69E, it appears that the monomer is active, albeit less so than the dimer, while for F52A, the monomer and dimer both appear to exhibit very low activity. The dimeric species is needed to obtain high specific activity. We conclude that, of the residues that were studied, Phe52 and Arg69 are the major determinants of dimer formation and a single mutation at either position substantially hinders dimerization. The use of a mutant glutathione S-transferase which retains activity yet has a greatly weakened tendency to dimerize (such as R69E) may be advantageous for certain applications of GST fusion proteins.


Subject(s)
Amino Acid Substitution , Glutathione Transferase/chemistry , Protein Subunits/chemistry , Amino Acid Substitution/genetics , Animals , Arginine/genetics , Circular Dichroism , Dimerization , Enzyme Activation/genetics , Enzyme Stability/genetics , Glutathione Transferase/biosynthesis , Glutathione Transferase/genetics , Glutathione Transferase/isolation & purification , Humans , Hydrophobic and Hydrophilic Interactions , Isoenzymes/biosynthesis , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/isolation & purification , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Phenylalanine/genetics , Protein Subunits/biosynthesis , Protein Subunits/genetics , Protein Subunits/isolation & purification , Rats , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Structural Homology, Protein
SELECTION OF CITATIONS
SEARCH DETAIL
...