scholarly journals Proton-linked l-rhamnose transport, and its comparison with l-fucose transport in Enterobacteriaceae

1993 ◽  
Vol 290 (3) ◽  
pp. 833-842 ◽  
Author(s):  
J A R Muiry ◽  
T C Gunn ◽  
T P McDonald ◽  
S A Bradley ◽  
C G Tate ◽  
...  
Keyword(s):  
Transport System ◽  
Erwinia Carotovora ◽  
Alkaline Ph ◽  
Transport Activity ◽  
Ph Change ◽  
Kinetic Measurements ◽  
E Coli ◽  
Steady State Kinetic ◽  
Competitive Inhibitors ◽  
State Kinetic

1. An alkaline pH change occurred when L-rhamnose, L-mannose or L-lyxose was added to L-rhamnose-grown energy-depleted suspensions of strains of Escherichia coli. This is diagnostic of sugar-H+ symport activity. 2. L-Rhamnose, L-mannose and L-lyxose were inducers of the sugar-H+ symport and of L-[14C]rhamnose transport activity. L-Rhamnose also induced the biochemically and genetically distinct L-fucose-H+ symport activity in strains competent for L-rhamnose metabolism. 3. Steady-state kinetic measurements showed that L-mannose and L-lyxose were competitive inhibitors (alternative substrates) for the L-rhamnose transport system, and that L-galactose and D-arabinose were competitive inhibitors (alternative substrates) for the L-fucose transport system. Additional measurements with other sugars of related structure defined the different substrate specificities of the two transport systems. 4. The relative rates of H+ symport and of sugar metabolism, and the relative values of their kinetic parameters, suggested that the physiological role of the transport activity was primarily for utilization of L-rhamnose, not for L-mannose or L-lyxose. 5. L-Rhamnose transport into subcellular vesicles of E. coli was dependent on respiration, was optimal at pH 7, and was inhibited by protonophores and ionophores. It was insensitive to N-ethylmaleimide or cytochalasin B. 6. L-Rhamnose, L-mannose and L-lyxose each elicited an alkaline pH change when added to energy-depleted suspensions of L-rhamnose-grown Salmonella typhimurium LT2, Klebsiella pneumoniae, Klebsiella aerogenes, Erwinia carotovora carotovora and Erwinia carotovora atroseptica. The relative rates of subsequent acidification varied, depending on both the organism and the sugar. L-Fucose promoted an alkaline pH change in all the L-rhamnose-induced organisms except the Erwinia species. No L-rhamnose-H+ symport occurred in any organism grown on L-fucose. 7. All these results showed that L-rhamnose transport into the micro-organisms occurred by a system different from that for L-fucose transport. Both systems are energized by the trans-membrane electrochemical gradient of protons. 8. Neither steady-state kinetic measurements nor binding-protein assays revealed the existence of a second L-rhamnose transport system in E. coli.

scholarly journals Proton-linked l-fucose transport in Escherichia coli

1987 ◽  
Vol 248 (2) ◽  
pp. 495-500 ◽  
Author(s):  
S A Bradley ◽  
C R Tinsley ◽  
J A R Muiry ◽  
P J F Henderson
Keyword(s):  
Escherichia Coli ◽  
Alkaline Ph ◽  
Electrochemical Gradient ◽  
Ph Change ◽  
Ph Optimum ◽  
Kinetic Measurements ◽  
E Coli ◽  
Steady State Kinetic ◽  
Periplasmic Proteins ◽  
State Kinetic

1. Addition of L-fucose to energy-depleted anaerobic suspensions of Escherichia coli elicited an uncoupler-sensitive alkaline pH change diagnostic of L-fucose/H+ symport activity. 2. L-Galactose or D-arabinose were also substrates, but not inducers, for the L-fucose/H+ symporter. 3. L-Fucose transport into subcellular vesicles was dependent upon respiration, displayed a pH optimum of about 5.5, and was inhibited by protonophores and ionophores. 4. These results showed that L-fucose transport into E. coli was energized by the transmembrane electrochemical gradient of protons. 5. Neither steady state kinetic measurements nor assays of L-fucose binding to periplasmic proteins revealed the existence of a second L-fucose transport system.

scholarly journals Dihydrolipoamide dehydrogenase from halophilic archaebacteria

1984 ◽  
Vol 218 (3) ◽  
pp. 811-818 ◽  
Author(s):  
M J Danson ◽  
R Eisenthal ◽  
S Hall ◽  
S R Kessell ◽  
D L Williams
Keyword(s):  
Steady State ◽  
Iodoacetic Acid ◽  
Enzymic Activity ◽  
Evolutionary Significance ◽  
Dihydrolipoamide Dehydrogenase ◽  
Kinetic Measurements ◽  
Steady State Kinetic ◽  
Halophilic Archaebacteria ◽  
Eukaryotic Organisms ◽  
State Kinetic

Dihydrolipoamide dehydrogenase has been discovered in the halophilic archaebacteria for the first time. The enzyme from both classical and alkaliphilic halobacteria has been investigated. (1) The enzyme specifically catalysed the stoichiometric oxidation of dihydrolipoamide by NAD+. Enzymic activity was optimal at 2 M-NaCl and was remarkably resistant to thermal denaturation. (2) The relative molecular masses (Mr) of the native enzyme from the various species of halobacteria were determined to be within the range 112000-120000. (3) The enzyme exhibited a hyperbolic dependence of catalytic activity on both dihydrolipoamide and NAD+ concentrations. From these steady-state kinetic measurements the dissociation constant (Ks) of dihydrolipoamide was determined to be 57 (+/- 5) microM. (4) The enzyme was only susceptible to inactivation by iodoacetic acid in the presence of its reducing ligands, dihydrolipoamide or NADH. The rate of inactivation followed a hyperbolic dependence on the concentration of dihydrolipoamide, from which the Ks of this substrate was calculated to be 55 (+/- 7) microM. Together with the steady-state kinetic data, the pattern of inactivations is consistent with the involvement in catalysis of a reversibly reducible disulphide bond, as has been found in dihydrolipoamide dehydrogenase from non-archaebacterial species. In eubacterial and eukaryotic organisms, dihydrolipoamide dehydrogenase functions in the 2-oxo acid dehydrogenase complexes. These multienzyme systems have not been detected in the archaebacteria, and, in the context of this apparent absence, the possible function and evolutionary significance of archaebacterial dihydrolipoamide dehydrogenase are discussed.

scholarly journals Energization of the transport systems for arabinose and comparison with galactose transport in Escherichia coli

1981 ◽  
Vol 200 (3) ◽  
pp. 611-627 ◽  
Author(s):  
K R Daruwalla ◽  
A T Paxton ◽  
P J Henderson
Keyword(s):  
Escherichia Coli ◽  
Transport System ◽  
Membrane Vesicles ◽  
Kinetic Studies ◽  
Transport Systems ◽  
Protonmotive Force ◽  
Alkaline Ph ◽  
Ph Change ◽  
Intact Cells ◽  
Membrane Bound

1. Strains of Escherichia coli were obtained containing either the AraE or the AraF transport system for arabinose. AraE+,AraF- strains effected energized accumulation and displayed an arabinose-evoked alkaline pH change indicative of arabinose-H+ symport. In contrast, AraE-,AraF+ strains accumulated arabinose but did not display H+ symport. 2. The ability of different sugars and their derivatives to elicit sugar-H+ symport in AraE+ strains was examined. Only L-arabinose and D-fucose were good substrates, and arabinose was the only inducer. 3. Membrane vesicles prepared from an AraE+,AraF+ strain accumulated the sugar, energized most efficiently by the respiratory substrates ascorbate + phenazine methosulphate. Addition of arabinose or fucose to an anaerobic suspension of membrane vesicles caused an alkaline pH change indicative or sugar-H+ symport on the membrane-bound transport system. 4. Kinetic studies and the effects of arsenate and uncoupling agents in intact cells and membrane vesicles gave further evidence that AraE is a low-affinity membrane-bound sugar-H+ symport system and that AraF is a binding-protein-dependent high-affinity system that does not require a transmembrane protonmotive force for energization. 5. The interpretation of these results is that arabinose transport into E. coli is energized by an electrochemical gradient of protons (AraE system) or by phosphate bond energy (AraF system). 6. In batch cultures the rates of growth and carbon cell yields on arabinose were lower in AraE-,AraF+ strains than in AraE+,AraF- or AraE+,AraF+ strains. The AraF system was more susceptible to catabolite repression than was the AraE system. 7. The properties of the two transport systems for arabinose are compared with those of the genetically and biochemically distinct transport systems for galactose, GalP and MglP. It appears that AraE is analogous to GalP, and AraF to MglP.

The stimulation by phosphate and the inhibition by adenine nucleotides and nicotinamide–adenine dinucleotide of the respiratory chain

1968 ◽  
Vol 46 (4) ◽  
pp. 315-321 ◽  
Author(s):  
M. C Blanchaer
Keyword(s):  
Respiratory Chain ◽  
Electron Acceptor ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nadh Dehydrogenase ◽  
Adenine Nucleotides ◽  
Coenzyme Q ◽  
Steady State Kinetic ◽  
Competitive Inhibitors ◽  
Energy Conserving ◽  
State Kinetic

Steady-state kinetic experiments with pigeon heart mitochondria and electron-transport particles (ETPH), with NADH as substrate in the absence of phosphate and with coenzyme Q1 or ferricyanide as the electron acceptor showed that ADP and ATP are competitive inhibitors at the NADH dehydrogenase level of the respiratory chain. The ADP effect was hyperbolic competitive in type indicating that this nucleotide inhibits by decreasing the affinity of the dehydrogenase for NADH rather than by competing for the NADH binding site of the enzyme. The inhibition by ADP and ATP does not involve energy-conserving reactions since it is not relieved by oligomycin or Dicoumarol. NAD+ also is an inhibitor of the NADH dehydrogenase of ETPH but is noncompetitive with respect to NADH. Phosphate stimulates NADH oxidation by ETPH and mitochondria in the absence of ADP when O2 is the electron acceptor. Since this effect is absent when CoQ1 or ferricyanide replaces O2 as the acceptor, it would appear that phosphate stimulates the respiratory chain on the O2 side of ubiquinone during noncoupled respiration.

Detection and characterization of VIM-52, a new variant of VIM-1 from Klebsiella pneumoniae clinical isolate

2021 ◽  
Author(s):  
Marie de Barsy ◽  
Paola Sandra Mercuri ◽  
Saoussen Oueslati ◽  
Eddy Elisée ◽  
Te-Din Huang ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Substrate Specificity ◽  
Clinical Isolate ◽  
Targeted Mutagenesis ◽  
Kinetic Measurements ◽  
Steady State Kinetic ◽  
New Variant ◽  
State Kinetic ◽  
Global Health Problem

Over the last two decades, antimicrobial resistance has become a global health problem. In Gram-negative bacteria, metallo-β-lactamases (MBLs), which inactivate virtually all β-lactams, increasingly contribute to this phenomenon. The aim of this study is to characterize VIM-52, a His224Arg variant of VIM-1, identified in a Klebsiella pneumoniae clinical isolate. VIM-52 conferred lower MICs to cefepime and ceftazidime as compared to VIM-1. These results were confirmed by steady state kinetic measurements, where VIM-52 yielded a lower activity towards ceftazidime and cefepime but not against carbapenems. Residue 224 is part of the L10 loop (residues 221-241), which borders the active site. As Arg 224 and Ser 228 are both playing an important and interrelated role in enzymatic activity, stability and substrate specificity for the MBLs, targeted mutagenesis at both positions were performed and further confirmed their crucial role for substrate specificity.

scholarly journals Inhibition of dimeric dihydrodiol dehydrogenases of rabbit and pig lens by ascorbic acid

1991 ◽  
Vol 275 (1) ◽  
pp. 121-126 ◽  
Author(s):  
A Hara ◽  
M Shinoda ◽  
T Kanazu ◽  
T Nakayama ◽  
Y Deyashiki ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Reverse Reaction ◽  
Ascorbate Oxidase ◽  
Binary Complex ◽  
Kinetic Measurements ◽  
Isoascorbic Acid ◽  
Dead End ◽  
Steady State Kinetic ◽  
Ic50 Values ◽  
State Kinetic

The dehydrogenase activity of dimeric dihydrodiol dehydrogenases (DD) purified from pig and rabbit lenses was inhibited by either L-ascorbic acid or its epimer, isoascorbic acid, at pH 7.5. Isoascorbate [IC50 (concn. giving 50% inhibition) = 0.043 mM for the pig enzyme; IC50 = 0.13 mM for the rabbit enzyme] was a more potent inhibitor than ascorbate (IC50 values 0.45 and 0.90 mM respectively), but 1 mM-dehydroascorbate gave less than 30% inhibition. Glucose, glucuronate, gulono-gamma-lactone, glutathione and dithiothreitol did not inhibit the enzyme activity. The inhibition by isoascorbate and ascorbate was instantaneous and reversible, and their inhibitory potency was decreased by addition of ascorbate oxidase. In the reverse reaction, isoascorbate and ascorbate gave low IC50 values of 0.013 and 0.10 mM respectively for the pig enzyme and 0.025 and 0.25 mM for the rabbit enzyme. The inhibition patterns by the two compounds were competitive with respect to dihydrodiols of naphthalene and benzene and uncompetitive with respect to NADP+, but those in the reverse reaction were uncompetitive with respect to both carbonyl substrate and NADPH. The steady-state kinetic measurements in the forward and reverse reactions by the pig enzyme were consistent with an ordered Bi Bi mechanism, in which NADP+ binds to the enzyme first and NADPH leaves last. The results indicate that ascorbate and its epimer directly bind to an enzyme: NADP+ binary complex as dead-end inhibitors. Thus ascorbate may be an important modulator of DD in the lens.

scholarly journals Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase

1999 ◽  
Vol 343 (2) ◽  
pp. 397-402 ◽  
Author(s):  
Daniel M. STOUGHTON ◽  
Gerardo ZAPATA ◽  
Robert PICONE ◽  
Willie F. VANN
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Active Site ◽  
Positive Charge ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
E Coli ◽  
Steady State Kinetic ◽  
State Kinetic ◽  
Escherichia Coli K1

Escherichia coli K1 CMP-sialic acid synthetase catalyses the synthesis of CMP-sialic acid from CTP and sialic acid. The active site of the 418 amino acid E. coli enzyme was localized to its N-terminal half. The bacterial CMP-sialic acid synthetase enzymes have a conserved motif, IAIIPARXXSKGLXXKN, at their N-termini. Several basic residues have been identified at or near the active site of the E. coli enzyme by chemical modification and site-directed mutagenesis. Only one of the lysines in the N-terminal motif, Lys-21, appears to be essential for activity. Mutation of Lys-21 in the N-terminal motif results in an inactive enzyme. Furthermore, Arg-12 of the N-terminal motif appears to be an active-site residue, based on the following evidence. Substituting Arg-12 with glycine or alanine resulted in inactive enzymes, indicating that this residue is required for enzymic activity. The Arg-12 → Lys mutant was partially active, demonstrating that a positive charge is required at this site. Steady-state kinetic analysis reveals changes in kcat, Km and Ks for CTP, which implicates Arg-12 in catalysis and substrate binding.

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