Characterization of the Elongating α-d-Mannosyl Phosphate Transferase from Three Species ofLeishmaniaUsing Synthetic Acceptor Substrate Analogues†

Methionine gamma-lyase (EC 4.4.1.11) was purified to homogeneity from the anaerobic protozoan parasite Trichomonas vaginalis by a series of f.p.l.c. procedures. The enzyme catalyses alpha gamma- and alpha beta-elimination reactions of a number of derivatives of methionine and cysteine. It also catalyses gamma-replacement reactions of the thiomethyl group of methionine, homocysteine and ethionine to yield the corresponding S-substituted homocysteine derivative. The enzyme is pyridoxal 5′-phosphate-dependent, has a native molecular mass of approx. 160 kDa and consists of four apparently identical subunits of molecular mass 43-45 kDa. The absorption spectrum of the enzyme is typical of those obtained for other pyridoxal 5′-phosphate-dependent enzymes, and the holoenzyme can be resolved to the apoenzyme by incubation with hydroxylamine and reconstituted by addition of the cofactor. The enzyme activity is significantly affected by carbonyl and thiol reagents, is competitively inhibited by a number of substrate analogues and is completely inactivated by the suicide inhibitor DL-propargylglycine. The T. vaginalis enzyme is similar, in terms of activity and properties, to the enzymes found in a number of species of bacteria that metabolize methionine under anaerobic conditions. It is suggested that methionine catabolism may be of particular importance to the survival of T. vaginalis under microaerophilic conditions in its host.

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Differential Inhibition of Cytosolic PEPCK by Substrate Analogues. Kinetic and Structural Characterization of Inhibitor Recognition‡

Biochemistry ◽

10.1021/bi7020662 ◽

2008 ◽

Vol 47 (7) ◽

pp. 2099-2109 ◽

Cited By ~ 21

Author(s):

Rose Mary Stiffin ◽

Sarah M. Sullivan ◽

Gerald M. Carlson ◽

Todd Holyoak

Keyword(s):

Structural Characterization ◽

Differential Inhibition ◽

Substrate Analogues ◽

Inhibitor Recognition

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Characterization of cytochrome bo3 activity in a native-like surface-tethered membrane

Biochemical Journal ◽

10.1042/bj20081345 ◽

2008 ◽

Vol 417 (2) ◽

pp. 555-560 ◽

Cited By ~ 20

Author(s):

Sophie A. Weiss ◽

Richard J. Bushby ◽

Stephen D. Evans ◽

Peter J. F. Henderson ◽

Lars J. C. Jeuken

Keyword(s):

Self Assembly ◽

Membrane System ◽

Terminal Oxidase ◽

E Coli ◽

Cytochrome Bo3 ◽

Substrate Analogues ◽

Long Chain ◽

Good Agreement ◽

Tethered Membranes

We have developed a simple native-like surface-tethered membrane system to investigate the activity of cbo3 (cytochrome bo3), a terminal oxidase in Escherichia coli. The tethered membranes consist of E. coli inner-membrane extracts mixed with additional E. coli lipids containing various amounts of the cbo3 substrate UQ-10 (ubiquinol-10). Tethered membranes are formed by self-assembly from vesicles on to gold electrodes functionalized with cholesterol derivatives. cbo3 activity was monitored using CV (cyclic voltammetry) with electron transfer to cbo3 mediated by UQ-10. The apparent Km for oxygen with this system is 1.1±0.4 μM, in good agreement with values reported in the literature for whole-cell experiments and for purified cbo3. Increasing the concentration of lipophilic UQ-10 in the membrane leads to an increase in cbo3 activity. The activity of cbo3 with long-chain ubiquinones appears to be different from previous reports using short-chain substrate analogues such as UQ-1 in that typical Michaelis–Menten kinetics are not observed using UQ-10. This native-like membrane model thus provides new insights into the interaction of transmembrane enzymes with hydrophobic substrates which contrasts with studies using hydrophilic UQ analogues.

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Structural Characterization of Early Michaelis Complexes in the Reaction Catalyzed by (+)-Limonene Synthase from Citrus sinensis Using Fluorinated Substrate Analogues

Biochemistry ◽

10.1021/acs.biochem.7b00144 ◽

2017 ◽

Vol 56 (12) ◽

pp. 1716-1725 ◽

Cited By ~ 16

Author(s):

Ramasamy P. Kumar ◽

Benjamin R. Morehouse ◽

Jason O. Matos ◽

Karan Malik ◽

Hongkun Lin ◽

...

Keyword(s):

Structural Characterization ◽

Citrus Sinensis ◽

Limonene Synthase ◽

Substrate Analogues

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A new phospholipase A2 inhibitor, unrelated to substrate analogues: kinetic characterization of the inhibition of secretory phospholipases A2 by PMS 832.

Journal of Lipid Mediators and Cell Signalling ◽

10.1016/s0929-7855(97)00008-4 ◽

1997 ◽

Vol 16 (3) ◽

pp. 171-187 ◽

Cited By ~ 2

Author(s):

Carine Binisti ◽

Carine Mounier ◽

Estera Touboul ◽

Françoise Heymans ◽

Cassian Bon ◽

...

Keyword(s):

Phospholipase A2 ◽

Kinetic Characterization ◽

Phospholipases A2 ◽

Substrate Analogues ◽

Phospholipase A2 Inhibitor

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Characterization of two partially purified xyloglucan endotransglycosylases from parsley (Petroselinum crispum) roots

Biologia ◽

10.2478/s11756-008-0067-2 ◽

2008 ◽

Vol 63 (3) ◽

Cited By ~ 4

Author(s):

Soňa Garajová ◽

Dana Flodrová ◽

Fairouz Ait-Mohand ◽

Vladimír Farkaš ◽

Eva Stratilová

Keyword(s):

Petroselinum Crispum ◽

Acidic Ph ◽

Xyloglucan Endotransglycosylase ◽

Acceptor Substrate ◽

Protein Mixture ◽

Donor Substrate ◽

Isoelectric Points ◽

Specific Activities

AbstractTwo forms of xyloglucan endotransglycosylase differing in isoelectric points were isolated from the protein mixture obtained from parsley roots and partially characterized. Both forms were glycoproteins differing in their specific activities but other features were almost the same. Activity and stability of both enzymes in broad pH region were observed with two pH optima, one at acidic pH (5.8) and the second one at basic pH (8.8). The enzymes behaved as typical transglycosylases since no activity was observed in the absence of xyloglucan oligosaccharides in the viscometric assay. Small hetero-transglycosylating activities were observed when hydroxyethyl-or carboxymethyl-celluloses instead of xyloglucan as donor substrate were used as well as when cello-oligosaccharides instead of xyloglucan oligosaccharides were used as the acceptor substrate.

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Expression and biochemical characterization of human glucose-6- phosphate dehydrogenase in Escherichia coli: a system to analyze normal and mutant enzymes

Blood ◽

10.1182/blood.v83.5.1436.bloodjournal8351436 ◽

1994 ◽

Vol 83 (5) ◽

pp. 1436-1441 ◽

Cited By ~ 1

Author(s):

TK Tang ◽

CH Yeh ◽

CS Huang ◽

MJ Huang

Keyword(s):

Escherichia Coli ◽

Biochemical Characterization ◽

Fold Increase ◽

Binding Domain ◽

Occurrence Rate ◽

Substrate Analogues ◽

Normal Enzyme ◽

Glucose 6 Phosphate Dehydrogenase

We have developed a system to characterize normal and mutated glucose-6- phosphate dehydrogenase (G6PD) enzymes in vitro. Normal or mutant G6PD cDNA was subcloned into a pGEX-3X vector, which allowed production of a functional fusion protein in Escherichia coli. When we compared the recombinant normal enzyme with authentic human G6PD, indistinguishable Km values for glucose-6-phosphate (G6P) and NADP were obtained, and the utilization rates for two substrate analogues (2-deoxy G6P and deamino NADP) also showed no difference between the enzymes. This system was used to assay a biochemically uncharacterized variant, G6PD Taipei (493 A-->wG; 165 Asn-->Asp), plus two other known mutations (487 G-->A; 163 Gly-->Ser and 592 C-->T; 198 Arg-->Cys) that are located close to or within the putative G6P binding domain. Our results show that the G6PD activities of these three mutants were greatly reduced. No significant alteration in G6PD kinetics was observed for both 487 and 493 mutations. However, a drastic reduction in the Km for G6P (4-fold decrease) and tremendous increases in utilization rates of 2-deoxy G6P (32-fold increase) and deamino NADP (6-fold increase) were associated with the 592 mutation. This results suggests that arginine 198 in human G6PD, possibly located within the putative G6P binding domain, may play an important role in binding the substrate G6P. In addition, we and others have recently identified that at least nine different types of mutations are responsible for G6PD deficiency in Chinese. In this report, we also present the occurrence rate of each mutation present in the population of Taiwan.

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Truncation of poly-N-acetylglucosamine (PNAG) polymerization with N-acetylglucosamine analogues

10.1101/2021.10.13.464265 ◽

2021 ◽

Author(s):

Zachary Morrison ◽

Alexander Eddenden ◽

Adithya S Subramanian ◽

P. Lynne Howell ◽

mark nitz

Keyword(s):

Biofilm Formation ◽

Chain Termination ◽

Salvage Pathway ◽

Biofilm Inhibition ◽

E Coli ◽

Substrate Analogues

Bacteria require polysaccharides for structure, survival, and virulence. Despite the central role these structures play in microbiology few tools are available to manipulate their production. In E. coli the glycosyltransferase complex PgaCD produces poly-N-acetylglucosamine (PNAG), an extracellular matrix polysaccharide required for biofilm formation. We report that C6-substituted (H, F, N3, SH, NH2) UDP-GlcNAc substrate analogues are inhibitors of PgaCD. In vitro the inhibitors cause PNAG chain termination; consistent with the mechanism of PNAG polymerization from the non-reducing terminus. In vivo, expression of the GlcNAc-1-kinase NahK in E. coli provided a non-native GlcNAc salvage pathway that produced the UDP-GlcNAc analogue inhibitors in situ. The 6-fluoro and 6-deoxy derivatives were potent inhibitors of biofilm formation in the transformed strain, providing a tool to manipulate this key exopolysaccharide. Characterization of the UDP-GlcNAc pool and quantification of PNAG generation support PNAG termination as the primary in vivo mechanism of biofilm inhibition by 6-fluoro UDP-GlcNAc.

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Spectroscopic and kinetic characterization of the light-dependent enzyme protochlorophyllide oxidoreductase (POR) using monovinyl and divinyl substrates

Biochemical Journal ◽

10.1042/bj20051635 ◽

2006 ◽

Vol 394 (1) ◽

pp. 243-248 ◽

Cited By ~ 14

Author(s):

Derren J. Heyes ◽

Jerzy Kruk ◽

C. Neil Hunter

Keyword(s):

Rhodobacter Capsulatus ◽

Chlorophyll Biosynthesis ◽

Kinetic Properties ◽

Biosynthesis Pathway ◽

Kinetic Characterization ◽

Protochlorophyllide Oxidoreductase ◽

Substrate Analogues ◽

Steady State Kinetic ◽

State Kinetic

The enzyme POR [Pchlide (protochlorophyllide) oxidoreductase] catalyses the reduction of Pchlide to chlorophyllide, which is a key step in the chlorophyll biosynthesis pathway. This light-dependent reaction has previously been studied in great detail but recent reports suggest that a mixture of MV (monovinyl) and DV (divinyl) Pchlides may have influenced some of these properties of the reaction. Low-temperature absorbance and fluorescence spectroscopy have revealed several spectral differences between MV and DV Pchlides, which were purified from a Rhodobacter capsulatus strain that was shown to contain a mixture of the two pigments. A thorough steady-state kinetic characterization using both Pchlide forms demonstrates that neither pigment appears to affect the kinetic properties of the enzyme. The reaction has also been monitored following illumination at low temperatures and was shown to consist of an initial photochemical step followed by four ‘dark’ steps for both pigments. However, minor differences were observed in the spectral properties of some of the intermediates, although the temperature dependency of each step was nearly identical for the two pigments. This work provides the first detailed kinetic and spectroscopic study of this unique enzyme using biologically important MV and DV substrate analogues. It also has significant implications for the DV reductase enzyme, which is responsible for converting DV pigments into their MV counterparts, and its position in the sequence of reactions that comprise the chlorophyll biosynthesis pathway.

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