The ATPase complex of Escherichia coli

1984 ◽  
Vol 62 (11) ◽  
pp. 1190-1197 ◽  
Author(s):  
Philip D. Bragg
Keyword(s):  
Escherichia Coli ◽  
Chemical Modification ◽  
Active Site ◽  
Active Sites ◽  
Transmembrane Protein ◽  
Proton Translocation ◽  
Amino Terminal ◽  
Loop Region ◽  
Cytoplasmic Surface ◽  
Hydrolysis Of

The ATPase (ATP synthase) complex of Escherichia coli is composed of an extrinsic membrane protein (ECF1), which contains the active site for ATP formation and hydrolysis, and is attached to ECF0, a transmembrane protein through which protons move to or from the active site on ECF1. ECF1 is composed of five subunits (α–ε) with a stoichiometry of α3β3γδε. The stoichiometry of the three subunits (a–c) of ECF0 is probably a1b2c10–15. In addition to 3 mol tightly bound adenine nucleotide/mol ECF1, three other "exchangeable" nucleotide binding sites can be detected. These sites are still present in the α and β subunit defective ECF1 of uncA401 and uncD412 mutants, although some changes in the tightness of binding are evident. The active sites of ECF1 require normal a and p subunits and may be present at αβ subunit interfaces. Hydrolysis of ATP requires cooperative interactions between α and β subunits. At low concentrations of ATP, in the absence of added divalent cations, hydrolysis of this substrate can occur at a single site without release of the product. This is consistent with alternating or sequential site mechanisms for ATP hydrolysis or synthesis. Predictions of secondary and tertiary structures from the known primary amino acid sequences of polypeptides a, b, and c have led to the following conclusions. Polypeptide a forms six or seven transmembrane a helices. The amino-terminal sequence of polypeptide b spans the membrane, but most of the protein is exposed on the cytoplasmic surface of the membrane where it can be cleaved by proteases in vitro. Polypeptide c consists of two nonpolar membrane-spanning α helices linked by a polar segment at the cytoplasmic surface of the membrane. This loop region interacts with ECF1 or is close to the ECF1-binding site. This is shown by competition between ECF1 and antibody for binding to polypeptide c. Chemical modification of arginyl residues in the loop region of polypeptide c inhibits ECF1 binding. Protease cleavage of polypeptide b affects, but does not abolish, binding of ECF1 to ECF0. Presumably, polypeptide b interacts with ECF1 also. The individual roles of the ECF0 polypeptides in proton translocation are not clear. Mutants in any of the three polypeptides may be defective in proton translocation. However, mutant and chemical modification studies support a role for the polypeptide c oligomer in the transmembrane proton pathway.

Effect of Modifiers on the Hydrolysis of Basic and Neutral Peptides by Carboxypeptidase B

1975 ◽  
Vol 53 (7) ◽  
pp. 747-757 ◽  
Author(s):  
Graham J. Moore ◽  
N. Leo Benoiton
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Kinetic Behavior ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Carboxypeptidase B ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Basic Peptides ◽  
Hydrolysis Of

The initial rates of hydrolysis of Bz-Gly-Lys and Bz-Gly-Phe by carboxypeptidase B (CPB) are increased in the presence of the modifiers β-phenylpropionic acid, cyclohexanol, Bz-Gly, and Bz-Gly-Gly. The hydrolysis of the tripeptide Bz-Gly-Gly-Phe is also activated by Bz-Gly and Bz-Gly-Gly, but none of these modifiers activate the hydrolysis of Bz-Gly-Gly-Lys, Z-Leu-Ala-Phe, or Bz-Gly-phenyllactic acid by CPB. All modifiers except cyclohexanol display inhibitory modes of binding when present in high concentration.Examination of Lineweaver–Burk plots in the presence of fixed concentrations of Bz-Gly has shown that activation of the hydrolysis of neutral and basic peptides by CPB, as reflected in the values of the extrapolated parameters, Km(app) and keat, occurs by different mechanisms. For Bz-Gly-Gly-Phe, activation occurs because the enzyme–modifier complex has a higher affinity than the free enzyme for the substrate, whereas activation of the hydrolysis of Bz-Gly-Lys derives from an increase in the rate of breakdown of the enzyme–substrate complex to give products.Cyclohexanol differs from Bz-Gly and Bz-Gly-Gly in that it displays no inhibitory mode of binding with any of the substrates examined, activates only the hydrolysis of dipeptides by CPB, and has a greater effect on the hydrolysis of the basic dipeptide than on the neutral dipeptide. Moreover, when Bz-Gly-Lys is the substrate, cyclohexanol activates its hydrolysis by CPB by increasing both the enzyme–substrate binding affinity and the rate of the catalytic step, an effect different from that observed when Bz-Gly is the modifier.The anomalous kinetic behavior of CPB is remarkably similar to that of carboxypeptidase A, and is a good indication that both enzymes have very similar structures in and around their respective active sites. A binding site for activator molecules down the cleft of the active site is proposed for CPB to explain the observed kinetic behavior.

Interaction specificity between the chaperone and proteolytic components of the cyanobacterial Clp protease

2012 ◽  
Vol 446 (2) ◽  
pp. 311-320 ◽  
Author(s):  
Anders Tryggvesson ◽  
Frida M. Ståhlberg ◽  
Axel Mogk ◽  
Kornelius Zeth ◽  
Adrian K. Clarke
Keyword(s):  
Escherichia Coli ◽  
Active Sites ◽  
Terminal Sequence ◽  
Core Complex ◽  
Clp Protease ◽  
E Coli ◽  
Loop Region ◽  
N Terminus ◽  
Specific Association ◽  
The Stability

The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. In plant chloroplasts and cyanobacteria, the essential constitutive Clp protease consists of the Hsp100/ClpC chaperone partnering a proteolytic core of catalytic ClpP and noncatalytic ClpR subunits. In the present study, we have examined putative determinants conferring the highly specific association between ClpC and the ClpP3/R core from the model cyanobacterium Synechococcus elongatus. Two conserved sequences in the N-terminus of ClpR (tyrosine and proline motifs) and one in the N-terminus of ClpP3 (MPIG motif) were identified as being crucial for the ClpC–ClpP3/R association. These N-terminal domains also influence the stability of the ClpP3/R core complex itself. A unique C-terminal sequence was also found in plant and cyanobacterial ClpC orthologues just downstream of the P-loop region previously shown in Escherichia coli to be important for Hsp100 association to ClpP. This R motif in Synechococcus ClpC confers specificity for the ClpP3/R core and prevents association with E. coli ClpP; its removal from ClpC reverses this core specificity.

scholarly journals A comparison of the active site of maltase-glucoamylase from the brush border of rabbit small intestine and kidney by chemical modification studies

1991 ◽  
Vol 274 (2) ◽  
pp. 349-354 ◽  
Author(s):  
B Pereira ◽  
S Sivakami
Keyword(s):  
Chemical Modification ◽  
Brush Border ◽  
Active Site ◽  
Brush Border Membrane ◽  
Kidney Enzyme ◽  
Rabbit Intestine ◽  
Rabbit Small Intestine ◽  
Intestinal Enzyme ◽  
The One ◽  
Hydrolysis Of

The neutral maltase-glucoamylase complex has been purified to homogeneity from the brush-border membrane of rabbit intestine and kidney. Chemical modification of the amino acid side chains was carried out on the purified enzymes. Studies on the kidney enzyme revealed that tryptophan, histidine and cysteine were essential for both maltase and glucoamylase activities, whereas tryptophan, histidine and lysine were essential for the maltase and glucoamylase activities of the intestinal enzyme. Though there was no difference in the amino acids essential for the hydrolysis of maltose and starch by any one enzyme, starch hydrolysis seems to require two histidine residues instead of the one which is required for maltose hydrolysis. This appears to be true for both the intestinal and kidney enzymes.

Investigation of the Catalytic Properties of the Dysthrombin, Thrombin Quick

1979 ◽  
Author(s):  
R Henriksen ◽  
W Owen ◽  
M Nesheim ◽  
K Mann
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Fluorescence Enhancement ◽  
Catalytic Mechanism ◽  
Antithrombin Iii ◽  
Catalytic Properties ◽  
Inhibitor Binding ◽  
Equivalent Number ◽  
Hydrolysis Of ◽  
Aggregation Of Platelets

Thrombin Quick (TQ) may be isolated following treatment of Prothrombin Quick [Owen, et al, Mayo Clinic Proceedings, 53: 29-33, (1978)] with Taipan venom, phospholipid and ca2+. The clotting activity of TQ with fibrinogen is 1/200 that of nornar thrombin (T). The activation of Factors V and VIII, and the aggregation of platelets by TQ occurs with an effectiveness of about 1/50 that of thrombin. when incubated with antithrombin III, both T ad TQ fom inhibitor complexes as determined by dodecylsulfate gel electropheresis. Titration of T and TQ with the fluorescent inhibitor dansylarginine-4-ethylpiperidine amide indicates an equivalent number of active sites based on protein absorption at 280 nm. However, the two enzymes may be distinquished by the decreased fluorescence enhancement observed with TQ relative to T, indicating an increased polarity in the inhibitor binding site of TQ. With the substrate benzoylarginine ethylester, TQ has a Km = 4.5 × 10-5M and kcat= 6.93 compared to Km = 4.0 × 10-5M and kcat= 17.7 for T. This indicates that the defect in TQ esterase activity is in the catalytic mechanism itself and not in substrate binding. The rate of inhibition of TQ by diisopropylphosphofluoridate is decreased. Decreased acylation and deacylation rates for TQ relative to T are observed for hydrolysis of the active site titrant 4-methykl-umbelliferyl-p-guanidinobenzoate

scholarly journals Evolution of Aromatic β-Glucoside Utilization by Successive Mutational Steps in Escherichia coli

2014 ◽  
Vol 197 (4) ◽  
pp. 710-716 ◽  
Author(s):  
Parisa Zangoui ◽  
Kartika Vashishtha ◽  
Subramony Mahadevan
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Selection Pressure ◽  
Second Step ◽  
Content Type ◽  
Catabolic Activity ◽  
Nucleotide Insertion ◽  
Esculin Hydrolysis ◽  
Steady State Levels ◽  
Hydrolysis Of

ThebglAgene ofEscherichia coliencodes phospho-β-glucosidase A capable of hydrolyzing the plant-derived aromatic β-glucoside arbutin. We report that the sequential accumulation of mutations inbglAcan confer the ability to hydrolyze the related aromatic β-glucosides esculin and salicin in two steps. In the first step, esculin hydrolysis is achieved through the acquisition of a four-nucleotide insertion within the promoter of thebglAgene, resulting in enhanced steady-state levels of thebglAtranscript. In the second step, hydrolysis of salicin is achieved through the acquisition of a point mutation within thebglAstructural gene close to the active site without the loss of the original catabolic activity against arbutin. These studies underscore the ability of microorganisms to evolve additional metabolic capabilities by mutational modification of preexisting genetic systems under selection pressure, thereby expanding their repertoire of utilizable substrates.

scholarly journals Interaction of L-threo and L-erythro isomers of 3-fluoroglutamate with glutamate decarboxylase from Escherichia coli

1985 ◽  
Vol 229 (3) ◽  
pp. 675-678 ◽  
Author(s):  
A Vidal-Cros ◽  
M Gaudry ◽  
A Marquet
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Glutamate Decarboxylase ◽  
Fluorine Atom ◽  
Pyridoxal Phosphate ◽  
Succinic Semialdehyde ◽  
E Coli ◽  
Rigid Conformation ◽  
The Difference ◽  
Hydrolysis Of

L-threo-3-Fluoroglutamate and L-erythro-3-fluoroglutamate were tested with glutamate decarboxylase from Escherichia coli. Both isomers were substrates: the threo isomer was decarboxylated into optically active 4-amino-3-fluorobutyrate, whereas the erythro isomer lost the fluorine atom during the reaction, yielding succinic semialdehyde after hydrolysis of the unstable intermediate enamine. The difference between the two isomers demonstrates that the glutamic acid-pyridoxal phosphate Schiff base is present at the active site under a rigid conformation. Furthermore, although the erythro isomer lost the fluorine atom, yielding a reactive aminoacrylic acid in the active site, no irreversible inactivation of E. coli glutamate decarboxylase was observed.

scholarly journals Structure ofEscherichia coliGrx2 in complex with glutathione: a dual-function hybrid of glutaredoxin and glutathioneS-transferase

2014 ◽  
Vol 70 (7) ◽  
pp. 1907-1913 ◽  
Author(s):  
Jun Ye ◽  
S. Venkadesh Nadar ◽  
Jiaojiao Li ◽  
Barry P. Rosen
Keyword(s):  
Escherichia Coli ◽  
Electron Donor ◽  
Active Site ◽  
Active Sites ◽  
Catalytic Cycle ◽  
Dual Function ◽  
Active Site Residues ◽  
E Coli ◽  
Arsenate Reduction ◽  
Gst Activity

The structure of glutaredoxin 2 (Grx2) fromEscherichia colico-crystallized with glutathione (GSH) was solved at 1.60 Å resolution. The structure of a mutant with the active-site residues Cys9 and Cys12 changed to serine crystallized in the absence of glutathione was solved to 2.4 Å resolution. Grx2 has an N-terminal domain characteristic of glutaredoxins, and the overall structure is congruent with the structure of glutathioneS-transferases (GSTs). Purified Grx2 exhibited GST activity. Grx2, which is the physiological electron donor for arsenate reduction byE. coliArsC, was docked with ArsC. The docked structure could be fitted with GSH bridging the active sites of the two proteins. It is proposed that Grx2 is a novel Grx/GST hybrid that functions in two steps of the ArsC catalytic cycle: as a GST it catalyzes glutathionylation of the ArsC–As(V) intermediate and as a glutaredoxin it catalyzes deglutathionylation of the ArsC–As(III)–SG intermediate.

scholarly journals Evolutionary Expansion of the Amidohydrolase Superfamily in Bacteria in Response to the Synthetic Compounds Molinate and Diuron

2015 ◽  
Vol 81 (7) ◽  
pp. 2612-2624 ◽  
Author(s):  
Elena Sugrue ◽  
Nicholas J. Fraser ◽  
Davis H. Hopkins ◽  
Paul D. Carr ◽  
Jeevan L. Khurana ◽  
...  
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Active Sites ◽  
Evolutionary Transition ◽  
Functional Changes ◽  
Metal Ion Analysis ◽  
Amidohydrolase Superfamily ◽  
Kinetics Of ◽  
Hydrolysis Of

ABSTRACTThe amidohydrolase superfamily has remarkable functional diversity, with considerable structural and functional annotation of known sequences. In microbes, the recent evolution of several members of this family to catalyze the breakdown of environmental xenobiotics is not well understood. An evolutionary transition from binuclear to mononuclear metal ion coordination at the active sites of these enzymes could produce large functional changes such as those observed in nature, but there are few clear examples available to support this hypothesis. To investigate the role of binuclear-mononuclear active-site transitions in the evolution of new function in this superfamily, we have characterized two recently evolved enzymes that catalyze the hydrolysis of the synthetic herbicides molinate (MolA) and phenylurea (PuhB). In this work, the crystal structures, mutagenesis, metal ion analysis, and enzyme kinetics of both MolA and PuhB establish that these enzymes utilize a mononuclear active site. However, bioinformatics and structural comparisons reveal that the closest putative ancestor of these enzymes had a binuclear active site, indicating that a binuclear-mononuclear transition has occurred. These proteins may represent examples of evolution modifying the characteristics of existing catalysts to satisfy new requirements, specifically, metal ion rearrangement leading to large leaps in activity that would not otherwise be possible.

scholarly journals Structure of l-Xylulose-5-Phosphate 3-Epimerase (UlaE) from the Anaerobic l-Ascorbate Utilization Pathway of Escherichia coli: Identification of a Novel Phosphate Binding Motif within a TIM Barrel Fold

2008 ◽  
Vol 190 (24) ◽  
pp. 8137-8144 ◽  
Author(s):  
Rong Shi ◽  
Marco Pineda ◽  
Eunice Ajamian ◽  
Qizhi Cui ◽  
Allan Matte ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Active Site ◽  
Active Sites ◽  
Triosephosphate Isomerase ◽  
Pentose Phosphate ◽  
Binding Motif ◽  
Phosphate Binding ◽  
Activity Measurements ◽  
Tim Barrel

ABSTRACT Three catabolic enzymes, UlaD, UlaE, and UlaF, are involved in a pathway leading to fermentation of l-ascorbate under anaerobic conditions. UlaD catalyzes a β-keto acid decarboxylation reaction to produce l-xylulose-5-phosphate, which undergoes successive epimerization reactions with UlaE (l-xylulose-5-phosphate 3-epimerase) and UlaF (l-ribulose-5-phosphate 4-epimerase), yielding d-xylulose-5-phosphate, an intermediate in the pentose phosphate pathway. We describe here crystallographic studies of UlaE from Escherichia coli O157:H7 that complete the structural characterization of this pathway. UlaE has a triosephosphate isomerase (TIM) barrel fold and forms dimers. The active site is located at the C-terminal ends of the parallel β-strands. The enzyme binds Zn2+, which is coordinated by Glu155, Asp185, His211, and Glu251. We identified a phosphate-binding site formed by residues from the β1/α1 loop and α3′ helix in the N-terminal region. This site differs from the well-characterized phosphate-binding motif found in several TIM barrel superfamilies that is located at strands β7 and β8. The intrinsic flexibility of the active site region is reflected by two different conformations of loops forming part of the substrate-binding site. Based on computational docking of the l-xylulose 5-phosphate substrate to UlaE and structural similarities of the active site of this enzyme to the active sites of other epimerases, a metal-dependent epimerization mechanism for UlaE is proposed, and Glu155 and Glu251 are implicated as catalytic residues. Mutation and activity measurements for structurally equivalent residues in related epimerases supported this mechanistic proposal.

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