scholarly journals Inhibition of Inositol Phosphorylceramide Synthase by the Cyclic Peptide Aureobasidin A

2009 ◽  
Vol 53 (2) ◽  
pp. 496-504 ◽  
Author(s):  
Paul A. Aeed ◽  
Casey L. Young ◽  
Marek M. Nagiec ◽  
Åke P. Elhammer
Keyword(s):  
Cyclic Peptide ◽  
Time Dependent ◽  
Mutant Enzyme ◽  
Kinetic Properties ◽  
Dependent Manner ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Noncompetitive Inhibitor ◽  
Aureobasidin A ◽  
Comparison Of The Results

ABSTRACT By using a detergent-washed membrane preparation, the interaction of the fungal natural product inhibitor aureobasidin A (AbA) with inositol phosphorylceramide synthase (IPC synthase) was studied by kinetic analysis of wild-type and mutant enzyme-catalyzed reactions. AbA inhibited the wild-type enzyme from both Candida albicans and Saccharomyces cerevisiae in an irreversible, time-dependent manner, with apparent Ki values of 183 and 234 pM, respectively. Three synthetic chemistry-derived AbA derivatives, PHA-533179, PHA-556655, and PHA-556656, had affinities 4 to 5 orders of magnitude lower and were reversible inhibitors that competed with the donor substrate phosphatidylinositol (PI). AbA was a reversible, apparently noncompetitive inhibitor, with a Ki of 1.4 μM, of the IPC synthase from an AbA-resistant S. cerevisiae mutant. The Km values for both substrates (ceramide and PI) were similar when they interacted with the mutant and the wild-type enzymes. By contrast, the V max for the mutant enzyme was less than 10% of that for the wild-type enzyme. A comparison of the results obtained with AbA with those obtained with two other natural products inhibitors, rustmicin and khafrefungin, revealed that while rustmicin appeared to be a reversible, noncompetitive inhibitor of the wild-type enzyme, with a Ki of 16.0 nM, khafrefungin had the kinetic properties of a time-dependent inhibitor and an apparent Ki of 0.43 nM. An evaluation of the efficiencies of these compounds as inhibitors of the mutant enzyme revealed for both a drop in the apparent affinity for the enzyme of more than 2 orders of magnitude.

scholarly journals Dissociation of the tetrameric phosphoglycerate mutase from yeast by a mutation in the subunit contact region

1993 ◽  
Vol 295 (3) ◽  
pp. 743-748 ◽  
Author(s):  
M F White ◽  
L A Fothergill-Gilmore ◽  
S M Kelly ◽  
N C Price
Keyword(s):  
Contact Region ◽  
Chloride Concentration ◽  
Mutant Enzyme ◽  
Phosphoglycerate Mutase ◽  
Kinetic Properties ◽  
Wild Type ◽  
Guanidinium Chloride ◽  
Wild Type Enzyme ◽  
Tetrameric Structure ◽  
Subunit Contact

Phosphoglycerate mutases from different sources exhibit a variety of quaternary structures (tetramer, dimer and monomer). To perturb the tetrameric structure of yeast phosphoglycerate mutase we have prepared a mutant enzyme in which Lys-168 in the subunit-contact region has been replaced by proline. The K168P mutant enzyme undergoes dissociation to dimers at low concentrations; thus on lowering the concentration from 200 micrograms/ml to 5 micrograms/ml the proportion of tetramer falls from 85% to 53%. The tetrameric structure of the wild-type enzyme remains intact over this range of concentrations. The mutant enzyme has similar kinetic properties to the wild-type enzyme, with kcat. being reduced by 26%. Far-u.v. c.d. studies show that there has been a small loss of helical structure in the mutant. Compared with wild-type enzyme, the K168P mutant enzyme is slightly less stable towards proteolysis by trypsin, but significantly less stable towards denaturation by guanidinium chloride, with the midpoint concentration of guanidinium chloride some 50% lower. After denaturation, the mutant enzyme could regain activity and quaternary structure when the guanidinium chloride concentration was lowered to 0.05 M. The properties of the mutant enzyme are discussed in terms of other dimeric phosphoglycerate and bisphosphoglycerate mutases which contain proline at position 168.

scholarly journals A subunit interface mutant of yeast pyruvate kinase requires the allosteric activator fructose 1,6-bisphosphate for activity

1995 ◽  
Vol 310 (1) ◽  
pp. 117-123 ◽  
Author(s):  
R A Collins ◽  
T McNally ◽  
L A Fothergill-Gilmore ◽  
H Muirhead
Keyword(s):  
Pyruvate Kinase ◽  
Mutant Enzyme ◽  
Hill Coefficient ◽  
Kinetic Properties ◽  
Variant Form ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Kinase Gene ◽  
Fructose 1,6 Bisphosphate ◽  
Allosteric Properties

A variant form of yeast pyruvate kinase (EC 2.7.1.40) with Ser-384 mutated to proline has been engineered in order to study the allosteric properties of this enzyme. Both the mutant and wild-type enzymes were overexpressed in a strain of yeast in which the genomic copy of the pyruvate kinase gene had been disrupted by an insertion of the Ura3 gene. Both enzymes were purified to homogeneity and their kinetic properties characterized. The wild-type enzyme displays sigmoid kinetics with respect to phosphoenolpyruvate (PEP) concentration, and is activated by the allosteric effect fructose 1,6-bisphosphate with concomitant reduction in co-operativity. In contrast, the mutant was found to be dependent on the presence of the effector for catalytic activity and was inactive in its absence. The fully activated mutant enzyme had a kcat. 1.6 times greater than that of the wild-type enzyme. The mutation introduced into the enzyme is in an intersubunit contact which is known to be critical for the allosteric properties of the enzyme, and is far removed from the active site. The major effect of the mutation seems to be to stabilize the low-affinity T state of the apoenzyme, although kcat. is also affected. The S0.5 for PEP and S0.5 for ADP of the wild-type enzyme were 0.22 +/- 0.004 and 0.15 +/- 0.01 mM respectively (means +/- S.E.M.). In the activated mutant enzyme, these kinetic parameters increased to 0.67 +/- 0.03 and 0.43 +/- 0.03 mM respectively. The cooperativity between ADP-binding sites was altered in the mutant enzyme, with the Hill coefficient (h) for ADP increasing to 1.65 +/- 0.07 in the presence of the effector, compared with a value of 0.01 +/- 0.07 for the wild-type enzyme under the same conditions. CD spectroscopy revealed the secondary structure of the mutant enzyme to be little different from that of the wild-type enzyme, indicating that the two enzymes have similar secondary structures in solution. Precise tertiary and quaternary structures such as intersubunit and interdomain interactions may be modified. An improved purification procedure has been devised that allows large quantities of enzyme to be rapidly prepared.

scholarly journals The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2

1993 ◽  
Vol 291 (1) ◽  
pp. 89-94 ◽  
Author(s):  
P White ◽  
F D C Manson ◽  
C E Brunt ◽  
S K Chapman ◽  
G A Reid
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Cytochrome C ◽  
Kinetic Properties ◽  
Stopped Flow ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Cytochrome C Reductase ◽  
Flavocytochrome B2 ◽  
Cytochrome C Reduction

The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino acid sequence of this interdomain hinge is dramatically different in flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the difference in kinetic properties between the two enzymes. To probe the importance of the hinge, an interspecies hybrid enzyme has been constructed comprising the bulk of the S. cerevisiae enzyme but containing the H. anomala flavocytochrome b2 hinge. The kinetic properties of this ‘hinge-swap’ enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase as is evident from steady-state experiments with ferricyanide as acceptor (only 3-fold less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (2.5-fold slower than in wild-type enzyme). The major effect of the hinge-swap mutation is to lower dramatically the enzyme's effectiveness as a cytochrome c reductase; kcat. for cytochrome c reduction falls by more than 100-fold, from 207 +/- 10 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 1.62 +/- 0.41 s-1 in the mutant enzyme. This fall in cytochrome c reductase activity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the kcat. for haem reduction in the hinge-swap enzyme (measured by the stopped-flow method) has a value of 1.61 +/- 0.42 s-1, identical with the value for cytochrome c reduction and some 300-fold lower than the value for the wild-type enzyme. From these and other kinetic parameters, including kinetic isotope effects with [2-2H]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer between the two domains of flavocytochrome b2.

scholarly journals CLL-Derived Exosomes Function As Trojan Horses That Induce SMAD6-Dependent Apoptosis of Normal B-Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1734-1734
Author(s):  
Orit Uziel ◽  
Zinab Sarsur- Amer ◽  
Einat Beery ◽  
Pia Raanani ◽  
Uri Rozovski
Keyword(s):  
B Cells ◽  
Time Dependent ◽  
Dependent Manner ◽  
Environmental Resources ◽  
Type B ◽  
Wild Type ◽  
Cell Competition ◽  
Western Immunoblotting ◽  
Competition Theory

Studies from recent years unraveled the role of monocytes and T-cells in the pathogenesis of chronic lymphocytic leukemia (CLL). The role of other immune cells in the pathobiology of CLL is less known. Specifically, whether B-cells, the normal counterpart of CLL cells play a role in CLL is unknown. Nevertheless, since both CLL cells and wild type B-cells reside in lymphatic organs and travel in blood, they either share or compete over common environmental resources. According to the cell competition theory, a sensing mechanism measures the relative fitness of a cell and ensures the elimination of cells deemed to be less fit then their neighbors. Since constitutive activation of intracellular pathways protect CLL cells from apoptosis, the cell competition theory predicts that compared with normal B-cells these cells are sensed as "super fit" and B-cells, the less fit counterparts, are eliminated. Yet, what delivers this massage across a population of cells is unknown. Exosomes are nanosized particles that are secreted by various types of cells. Exosomes carry a cargo of proteins and different types of RNA. They travel in body fluids and are taken up by cells in their vicinity. Since cancer cells including CLL cells secrete exosomes, we have formulated our hypothesis, namely, that exosomes derived from CLL cells are the vehicles that carry a death massage to wild type B-cells. To test this hypothesis, we isolated CLL cells from 3 previously untreated patients with CLL. We then grew these cells in exosome free media for 72 hours and harvested the exosomes by ultracentrifugation. We used NanoSight tracking analysis, Western immunoblotting for CD63, a common exosomal marker, and electron microscopy imaging studies to ensure that our pellet include the typical 100nm exosomal particles. Subsequently, we subjected normal B-cells derived from healthy volunteers to CLL derived exosomes stained by FM-143 dye. Using flow cytometry we found that exosomes are taken up by normal B-cells in a dose- and time- dependent manner. Double staining of the recipient B-cells to Annexin/PI revealed that exosomes induce apoptosis of these cells in a dose- and time- dependent manner. We then used RNA-seq to trace the changes in the molecular makeup of B-cells after exosomal uptake?? they took up exosomes. We found 24 transcripts that were differentially expressed (11 that were upregulated and 13 that were downregulated). We then verified the array results by quantitative real-time PCR for four of these genes. Among the top transcripts that were upregulated in exosome-positive B-cells is SMAD6. Because the upregulation of the SMAD family members including SMAD6 is associated with the induction of apoptosis in various malignant and non-malignant cells we wondered whether the upregulation of SMAD6 also induces apoptosis in normal B-cells. To test this, we transfected normal B-cells with SMAD6 containing vector and verified by RT-PCR that level of SMAD6 transcript were upregulated and by Western immunoblotting that levels of SMAD6 protein are upregulated as well. As expected, the rate of apoptosis was higher, and the rates of viable cells and proliferating cells were significantly lower in SMAD6-transfected B-cells. Taken together, we show here that CLL cells secrete exosomes that function as "Trojan horses". Once they are taken up by normal B-cells they induce SMAD6-dependent apoptosis. In this way the neoplastic cells may actively eliminate their competitors and take over the common environmental resources. Disclosures No relevant conflicts of interest to declare.

scholarly journals Tyr-143 facilitates interdomain electron transfer in flavocytochrome b2

1992 ◽  
Vol 285 (1) ◽  
pp. 187-192 ◽  
Author(s):  
C S Miles ◽  
N Rouvière-Fourmy ◽  
F Lederer ◽  
F S Mathews ◽  
G A Reid ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Isotope Effects ◽  
Mutant Enzyme ◽  
Stopped Flow ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Flavocytochrome B2 ◽  
Rate Determining Step ◽  
Kinetic Isotope

The role of Tyr-143 in the catalytic cycle of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) has been examined by replacement of this residue with phenylalanine. The electron-transfer steps in wild-type and mutant flavocytochromes b2 have been investigated by using steady-state and stopped-flow kinetic methods. The most significant effect of the Tyr-143----Phe mutation is a change in the rate-determining step in the reduction of the enzyme. For wild-type enzyme the main rate-determining step is proton abstraction at the C-2 position of lactate, as shown by the 2H kinetic-isotope effect. However, for the mutant enzyme it is clear that the slowest step is interdomain electron transfer between the FMN and haem prosthetic groups. In fact, the rate of haem reduction by lactate, as determined by the stopped-flow method, is decreased by more than 20-fold, from 445 +/- 50 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 21 +/- 2 s-1 in the mutant enzyme. Decreases in kinetic-isotope effects seen with [2-2H]lactate for mutant enzyme compared with wild-type, both for flavin reduction (from 8.1 +/- 1.4 to 4.3 +/- 0.8) and for haem reduction (from 6.3 +/- 1.2 to 1.6 +/- 0.5) also provide support for a change in the nature of the rate-determining step. Other kinetic parameters determined by stopped-flow methods and with two external electron acceptors (cytochrome c and ferricyanide) under steady-state conditions are all consistent with this mutation having a dramatic effect on interdomain electron transfer. We conclude that Tyr-143, an active-site residue which lies between the flavodehydrogenase and cytochrome domains of flavocytochrome b2, plays a key role in facilitating electron transfer between FMN and haem groups.

scholarly journals Mutational Replacement of Leu-293 in the Class C Enterobacter cloacae P99 β-Lactamase Confers Increased MIC of Cefepime

2002 ◽  
Vol 46 (6) ◽  
pp. 1966-1970 ◽  
Author(s):  
Sergei B. Vakulenko ◽  
Dasantila Golemi ◽  
Bruce Geryk ◽  
Maxim Suvorov ◽  
James R. Knox ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Broad Spectrum ◽  
Random Mutagenesis ◽  
Enterobacter Cloacae ◽  
The Other ◽  
Mutant Enzyme ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Wide Range ◽  
Class C

ABSTRACT The class C β-lactamase from Enterobacter cloacae P99 confers resistance to a wide range of broad-spectrum β-lactams but not to the newer cephalosporin cefepime. Using PCR mutagenesis of the E. cloacae P99 ampC gene, we obtained a Leu-293-Pro mutant of the P99 β-lactamase conferring a higher MIC of cefepime (MIC, 8 μg/ml, compared with 0.5 μg/ml conferred by the wild-type enzyme). In addition, the mutant enzyme produced higher resistance to ceftazidime but not to the other β-lactams tested. Mutants with 15 other replacements of Leu-293 were prepared by site-directed random mutagenesis. None of these mutant enzymes conferred MICs of cefepime higher than that conferred by Leu-293-Pro. We determined the kinetic parameters of the purified E. cloacae P99 β-lactamase and the Leu-293-Pro mutant enzyme. The catalytic efficiencies (k cat/Km ) of the Leu-293-Pro mutant β-lactamase for cefepime and ceftazidime were increased relative to the respective catalytic efficiencies of the wild-type P99 β-lactamase. These differences likely contribute to the higher MICs of cefepime and ceftazidime conferred by this mutant β-lactamase.

scholarly journals Replacement of Tyrosine 181 by Phenylalanine in Gentisate 1,2-Dioxygenase I from Pseudomonas alcaligenes NCIMB 9867 Enhances Catalytic Activities

2005 ◽  
Vol 187 (21) ◽  
pp. 7543-7545 ◽  
Author(s):  
Chew Ling Tan ◽  
Chew Chieng Yeo ◽  
Hoon Eng Khoo ◽  
Chit Laa Poh
Keyword(s):  
Catalytic Efficiency ◽  
Relative Activity ◽  
Site Directed Mutagenesis ◽  
Mutant Enzyme ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Specific Mutation ◽  
Catalytic Activities ◽  
Pseudomonas Alcaligenes

ABSTRACT xlnE, encoding gentisate 1,2-dioxygenase (EC 1.13.11.4), from Pseudomonas alcaligenes (P25X) was mutagenized by site-directed mutagenesis. The mutant enzyme, Y181F, demonstrated 4-, 3-, 6-, and 16-fold increases in relative activity towards gentisate and 3-fluoro-, 4-methyl-, and 3-methylgentisate, respectively. The specific mutation conferred a 13-fold higher catalytic efficiency (k cat/Km ) on Y181F towards 3-methylgentisate than that of the wild-type enzyme.

scholarly journals Role of Arg-401 of cytosolic serine hydroxymethyltransferase in subunit assembly and interaction with the substrate carboxy group

1997 ◽  
Vol 327 (3) ◽  
pp. 877-882 ◽  
Author(s):  
Junutula Reddy JAGATH ◽  
Naropantul APPAJI RAO ◽  
Handanahal SubbaRao SAVITHRI
Keyword(s):  
Carboxy Group ◽  
Gel Filtration ◽  
Serine Hydroxymethyltransferase ◽  
Site Directed Mutagenesis ◽  
Mutant Enzyme ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Partial Dissociation ◽  
Tetrameric Form

In an attempt to identify the arginine residue involved in binding of the carboxylate group of serine to mammalian serine hydroxymethyltransferase, a highly conserved Arg-401 was mutated to Ala by site-directed mutagenesis. The mutant enzyme had a characteristic visible absorbance at 425 nm indicative of the presence of bound pyridoxal 5ʹ-phosphate as an internal aldimine with a lysine residue. However, it had only 0.003% of the catalytic activity of the wild-type enzyme. It was also unable to perform reactions with glycine, β-phenylserine or D-alanine, suggesting that the binding of these substrates to the mutant enzyme was affected. This was also evident from the interaction of amino-oxyacetic acid, which was very slow (8.4×10-4 s-1 at 50 μM) for the R401A mutant enzyme compared with the wild-type enzyme (44.6 s-1 at 50 μM). In contrast, methoxyamine (which lacks the carboxy group) reacted with the mutant enzyme (1.72 s-1 at 250 μM) more rapidly than the wild-type enzyme (0.2 s-1 at 250 μM). Further, both wild-type and the mutant enzymes were capable of forming unique quinonoid intermediates absorbing at 440 and 464 nm on interaction with thiosemicarbazide, which also does not have a carboxy group. These results implicate Arg-401 in the binding of the substrate carboxy group. In addition, gel-filtration profiles of the apoenzyme and the reconstituted holoenzyme of R401A and the wild-type enzyme showed that the mutant enzyme remained in a tetrameric form even when the cofactor had been removed. However, the wild-type enzyme underwent partial dissociation to a dimer, suggesting that the oligomeric structure was rendered more stable by the mutation of Arg-401. The increased stability of the mutant enzyme was also reflected in the higher apparent melting temperature (Tm) (61 °C) than that of the wild-type enzyme (56 °C). The addition of serine or serinamide did not change the apparent Tm of R401A mutant enzyme. These results suggest that the mutant enzyme might be in a permanently ‘open’ form and the increased apparent Tm could be due to enhanced subunit interactions.

Phosphoglycerate Kinase Deficiency: Characterization of the Wild-Type Enzyme and Three Pathological Variants Generated from C.140T>a, C.491A>T and C.959G>a Mutations

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2875-2875
Author(s):  
Simone Morera ◽  
Laurent Chiarelli ◽  
Stefano Rovida ◽  
Paola Bianchi ◽  
Elisa Fermo ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Clinical Manifestations ◽  
Phosphoglycerate Kinase ◽  
Free Form ◽  
Heat Inactivation ◽  
Kinetic Properties ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Terminal Domain

Abstract Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible transfer of a phoshoryl-group from 1,3-bisphosphoglycerate (1,3-BPG) to ADP forming 3-phosphoglycerate (3-PG) and ATP. PGK is a typical two-domain hinge-bending enzyme, with a highly conserved structure. The N-terminal domain binds 1,3-BPG/3-PG, whereas the C-terminal domain binds Mg-ADP/Mg-ATP.Humans have two PGK isozymes, PGK1 and PGK2, where PGK1 is an ubiquitous enzyme that is expressed in all somatic cells and PGK2 is a testis-specific enzyme. The PGK1 gene is located on the X-chromosome q-13.1, contains 11 exons and encodes a protein of 416 amino acids. Mutations of the PGK1 gene result in an enzyme deficiency that is for the most clinically characterized by mild-to severe hemolytic anemia and various defects in the central nervous system. To date, 19 different mutations with worldwide distribution have been reported. No correlation between the residual PGK activity and the severity of the clinical manifestations have been documented so far. To analyze the mutations at protein level and possibly to correlate the genotype to clinical phenotype, we started with the molecular characterization of the wild-type PGK1 enzyme and three mutants (I47N, D164 and S320N) obtained from E.coli as recombinant proteins. The corresponding mutations, i.e., c.140T>A, c.491A>T and c.959G>A, have been identified in patients with PGK deficiency and affected by severe hemolytic anemia and progressive mental retardation. The cDNA encoding the PGK1 was prepared starting from a blood sample of a healthy donor, with normal PGK1 activity. Site-directed mutagenesis was used to introduce the desired mutations into the PGK1 cDNA. The wild type enzyme was expressed to its maximum level (about 80–100 mg of enzyme per liter of culture) after 5 hours of induction with 0.5 mM IPTG at 37 °C. For mutant enzymes the induction temperature was lowered to 25°C. All recombinant enzymes were purified to homogeneity after a single chromatographic step on DEAE Sepharose column. The wild-type enzyme was crystallized in both free form or complexed with 3-PG. The corresponding structures were solved to high resolution (1.8 and 1.6 A, respectively) and compared. Essentially, binding 3-PG caused a 6° rotation of the N-domain in respect to the C-domain. The recombinant enzyme exhibited kinetic properties similar to those of the authentic enzyme, displaying vs 3-PG and ATP alike specific activities (about 1000 U/mg) and alike Km values (about 1mM). I47N and S320N mutant enzymes showed kcat values 3-fold lower than the wild-type enzyme. The D164V was characterized by a Km value vs 3-PG 15 times higher than that of the other enzymes studied and a catalytic efficiency 70 times lower. Finally, all mutant enzymes turned out to be highly heat unstable with respect to the wildtype enzyme, losing half of their activity after approximately 10 minutes of incubation at 37 °C. At higher temperatures, the wild-type enzyme was protected from heat inactivation by Mg-ATP or 3-PG. On the contrary, no one mutant was protect by Mg-ATP and the D164V and S320N mutants were not even protected by 3-PG. Therefore, these preliminary studies indicate that all mutations target amino acid residues located in positions primarily important for preserving the protein stability during the red cell life span.

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