scholarly journals Redox properties and cross-linking of the dithiol/disulphide active sites of mammalian protein disulphide-isomerase

1991 ◽  
Vol 275 (2) ◽  
pp. 341-348 ◽  
Author(s):  
H C Hawkins ◽  
M de Nardi ◽  
R B Freedman
Keyword(s):  
Redox Potential ◽  
Active Site ◽  
Active Sites ◽  
Residual Activity ◽  
Redox Properties ◽  
Redox Couple ◽  
Hill Coefficient ◽  
Cross Linking ◽  
Standard Redox Potential ◽  
Reactive Groups

1. The redox properties of the active-site dithiol/disulphide groups of PDI were determined by equilibrating the enzyme with an excess of GSH + GSSG, rapidly alkylating the dithiol form of the enzyme to inactivate it irreversibly, and determining the proportion of the disulphide form by measuring the residual activity under standard conditions. 2. The extent of reduction varied with the applied redox potential; to a first approximation, the data fitted a model in which all the enzyme dithiol/disulphide groups are independent and equivalent and the equilibrium constant between these sites and the GSH/GSSG redox couple is 42 microM at pH 7.5. 3. The standard redox potential for PDI active-site dithiol/disulphide couples was calculated from this result and found to be -0.11 V; hence PDI is a stronger oxidant and weaker reductant than GSH, nicotinamide cofactors, thioredoxin and dithiothreitol. 4. The redox equilibrium data for PDI with the GSH/GSSG redox couple showed sigmoidal deviations from linearity. The sigmoidicity could be modelled closely by assuming a Hill coefficient of 1.5. 5. This evidence of co-operative interactions between the four active sites in a PDI dimer was extended by studying the reaction between PDI and homobifunctional alkylating agents with various lengths between the reactive groups. A species whose electrophoretic mobility suggested it contained an intrachain cross-link was observed in all cases, whereas there was no evidence for cross-linking between the chains of the PDI homodimer. Most effective cross-linking was achieved with reagents containing five or more methylene spacer groups, implying a minimum distance of 1.6 nm (16 A) between the active-site reactive groups within the two thioredoxin-like domains of the PDI polypeptide.

scholarly journals Characterization of redox properties of FAD cofactor of Thermotoga maritima thioredoxin reductase

Chemija ◽  
2020 ◽  
Vol 31 (3) ◽  
Author(s):  
Benjaminas Valiauga ◽  
Nicolas Rouhier ◽  
Jean-Pierre Jacquot ◽  
Narimantas Čėnas
Keyword(s):  
Amino Acid ◽  
Redox Potential ◽  
Thermotoga Maritima ◽  
Thioredoxin Reductase ◽  
Amino Acid Sequences ◽  
Redox Properties ◽  
Redox Couple ◽  
E Coli ◽  
Standard Redox Potential

The fluorescence properties of FAD of Thermotoga maritima thioredoxin reductase (TmTR), taken together with the amino acid sequences and structures of similar TRs, are consistent with the interdomain rotation in the catalysis of TmTR. The standard redox potential of FAD of TmTR, –0.230 V, determined by the reactions with 3-acetylpyridine adenine dinucleotide (APAD+/APADH) redox couple, is close to that of E. coli TR. During the reduction of duroquinone with TmTR, the transient formation of neutral FAD semiquinone, and, possibly, FADH2–NAD+ complex was observed. This shows that in spite of obligatory twoelectron (hydride)-transfer between NADH and physiological disulfide oxidants, the FAD cofactor of TmTR may exist under a stable semiquinone form.

Photochemical mapping of the active site of myosin

1992 ◽  
Vol 336 (1276) ◽  
pp. 55-61 ◽  
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Active Sites ◽  
Cross Linking ◽  
Detailed Structure ◽  
Atp Binding Site ◽  
Adenine Ring ◽  
Skeletal Myosin ◽  
Key Residues ◽  
Specific Labelling

The active sites of myosin from skeletal, smooth and scallop muscle have been partly characterized by use of a series of photoreactive analogues of ATP. Specific labelling was attained by trapping these analogues in their diphosphate forms at the active sites by either cross-linking two reactive thiols (skeletal myosin) or by formation of stable vanadate-metal ion transition state-like complexes (smooth muscle and scallop myosin). By use of this approach combined with appropriate chemistry, several key residues in all three myosins have been identified which bind at or near the adenine ring, the ribose ring and to the γ-phosphate of ATP. This information should aid in the solution of the crystal structure of the heads of myosin and in defining a detailed structure of the ATP binding site.

scholarly journals Effect of Sequences of the Active-Site Dipeptides of DsbA and DsbC on In Vivo Folding of Multidisulfide Proteins inEscherichia coli

2001 ◽  
Vol 183 (3) ◽  
pp. 980-988 ◽  
Author(s):  
Paul H. Bessette ◽  
Ji Qiu ◽  
James C. A. Bardwell ◽  
James R. Swartz ◽  
George Georgiou
Keyword(s):  
Redox Potential ◽  
Active Site ◽  
Active Sites ◽  
Multicopy Plasmid ◽  
Redox Potentials ◽  
Cxxc Motif ◽  
Wide Range ◽  
Significant Difference ◽  
Disulfide Isomerization

ABSTRACT We have examined the role of the active-site CXXC central dipeptides of DsbA and DsbC in disulfide bond formation and isomerization in the Escherichia coli periplasm. DsbA active-site mutants with a wide range of redox potentials were expressed either from the trc promoter on a multicopy plasmid or from the endogenous dsbA promoter by integration of the respective alleles into the bacterial chromosome. ThedsbA alleles gave significant differences in the yield of active murine urokinase, a protein containing 12 disulfides, including some that significantly enhanced urokinase expression over that allowed by wild-type DsbA. No direct correlation between the in vitro redox potential of dsbA variants and the urokinase yield was observed. These results suggest that the active-site CXXC motif of DsbA can play an important role in determining the folding of multidisulfide proteins, in a way that is independent from DsbA's redox potential. However, under aerobic conditions, there was no significant difference among the DsbA mutants with respect to phenotypes depending on the oxidation of proteins with few disulfide bonds. The effect of active-site mutations in the CXXC motif of DsbC on disulfide isomerization in vivo was also examined. A library of DsbC expression plasmids with the active-site dipeptide randomized was screened for mutants that have increased disulfide isomerization activity. A number of DsbC mutants that showed enhanced expression of a variant of human tissue plasminogen activator as well as mouse urokinase were obtained. These DsbC mutants overwhelmingly contained an aromatic residue at the C-terminal position of the dipeptide, whereas the N-terminal residue was more diverse. Collectively, these data indicate that the active sites of the soluble thiol- disulfide oxidoreductases can be modulated to enhance disulfide isomerization and protein folding in the bacterial periplasmic space.

Structural and Redox Properties of the Leaderless DsbE (CcmG) Protein: Both Active-Site Cysteines of the Reduced Form Are Involved in Its Function in the Escherichia coli Periplasm

2001 ◽  
Vol 382 (12) ◽  
pp. 1679-1686 ◽  
Author(s):  
Qi Li ◽  
Hong-Yu Hu ◽  
Wei-Qing Wang ◽  
Gen-Jun Xu
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Active Site ◽  
Redox Reaction ◽  
Disulfide Bonds ◽  
Biochemical Properties ◽  
Redox Properties ◽  
Reduced Form ◽  
Cytochrome C Maturation ◽  
Standard Redox Potential

AbstractThe thiol/disulfide oxidoreductases play important roles in ensuring the correct formation of disulfide bonds, of which the DsbE protein, also called CcmG, is the one implicated in electron transfer for cytochrome c maturation in the periplasm of Escherichia coli. The soluble, Nterminally truncated DsbE was overexpressed and purified to homogeneity. Here we report the structural and redox properties of the leaderless form (DsbEL ). During the redox reaction, the protein undergoes a structural transformation resulting in a more stable reduced form, but this form shows very low reactivity in thiol/ disulfide exchange of cysteine residues and low activity in accelerating the reduction of insulin. The standard redox potential (E' 0 ) for the active thiol/ disulfide was determined to be 0.186 V; only one of the two cysteines (Cys80) was suggested to be the active residue in the redox reaction. From the aspect of biochemical properties, DsbE can be regarded as a weak reductant in the Escherichia coli periplasm. This implies that the function of DsbE in cytochrome c maturation can be ascribed to its active site cysteines and the structure of the reduced form.

scholarly journals Inhibitory Proteins Block Substrate Access to the Active Site of Bacillus subtilis Intramembrane Metalloprotease SpoIVFB

2021 ◽  
Author(s):  
Sandra Olenic ◽  
Lim Heo ◽  
Michael Feig ◽  
Lee Kroos
Keyword(s):  
Bacillus Subtilis ◽  
Active Site ◽  
Active Sites ◽  
Structural Model ◽  
Terminal Region ◽  
Cross Linking ◽  
Intramembrane Proteases ◽  
Inhibitory Proteins ◽  
Partial Homology ◽  
Active Site Region

Intramembrane proteases of diverse signaling pathways use membrane-embedded active sites to cleave membrane-associated substrates. Interactions of intramembrane metalloproteases with modulators are poorly understood. Inhibition of Bacillus subtilis intramembrane metalloprotease SpoIVFB requires BofA and SpoIVFA, which transiently prevent cleavage of Pro-σK during endosporulation. Three conserved BofA residues (N48, N61, T64) in or near predicted transmembrane segment (TMS) 2 were found to be required for SpoIVFB inhibition. Disulfide cross-linking indicated that BofA TMS2 occupies the SpoIVFB active site region. BofA and SpoIVFA neither prevented SpoIVFB from interacting with Pro-σK in co-purification assays nor interfered with cross-linking between the C-terminal regions of Pro-σK and SpoIVFB. However, BofA and SpoIVFA did interfere with cross-linking between the N-terminal Proregion of Pro-σK and the SpoIVFB active site region and interdomain linker. A BofA variant lacking predicted TMS1, in combination with SpoIVFA, was less effective at interfering with some of the cross-links and slightly less effective at inhibiting cleavage of Pro-σK by SpoIVFB. A structural model was built of SpoIVFB in complex with BofA and parts of SpoIVFA and Pro-σK, using partial homology and constraints from cross-linking and co-evolutionary analyses. The model predicts that N48 in BofA TMS2 interacts with T64 (and possibly N61) of BofA to stabilize a membrane-embedded C-terminal region. SpoIVFA is predicted to bridge the BofA C-terminal region and SpoIVFB. Thus, the two inhibitory proteins block access of the Pro-σK N-terminal region to the SpoIVFB active site region. Our findings may inform efforts to develop selective inhibitors of intramembrane metalloproteases.

Catalytic Resonance Theory: Parallel Reaction Pathway Control

2019 ◽  
Author(s):  
M. Alexander Ardagh ◽  
Manish Shetty ◽  
Anatoliy Kuznetsov ◽  
Qi Zhang ◽  
Phillip Christopher ◽  
...  
Keyword(s):  
Chemical Reactions ◽  
Active Site ◽  
Active Sites ◽  
Reaction Pathway ◽  
Control Strategies ◽  
Oscillation Amplitude ◽  
Catalytic Performance ◽  
Parallel Reaction ◽  
Resonance Theory ◽  
Static Conditions

Catalytic enhancement of chemical reactions via heterogeneous materials occurs through stabilization of transition states at designed active sites, but dramatically greater rate acceleration on that same active site is achieved when the surface intermediates oscillate in binding energy. The applied oscillation amplitude and frequency can accelerate reactions orders of magnitude above the catalytic rates of static systems, provided the active site dynamics are tuned to the natural frequencies of the surface chemistry. In this work, differences in the characteristics of parallel reactions are exploited via selective application of active site dynamics (0 < ΔU < 1.0 eV amplitude, 10<sup>-6</sup> < f < 10<sup>4</sup> Hz frequency) to control the extent of competing reactions occurring on the shared catalytic surface. Simulation of multiple parallel reaction systems with broad range of variation in chemical parameters revealed that parallel chemistries are highly tunable in selectivity between either pure product, even when specific products are not selectively produced under static conditions. Two mechanisms leading to dynamic selectivity control were identified: (i) surface thermodynamic control of one product species under strong binding conditions, or (ii) catalytic resonance of the kinetics of one reaction over the other. These dynamic parallel pathway control strategies applied to a host of chemical conditions indicate significant potential for improving the catalytic performance of many important industrial chemical reactions beyond their existing static performance.

scholarly journals Crystal Structure, Exogenous Ligand Binding, and Redox Properties of an Engineered Diiron Active Site in a Bacterial Hemerythrin

2013 ◽  
Vol 52 (22) ◽  
pp. 13014-13020 ◽  
Author(s):  
Yasunori Okamoto ◽  
Akira Onoda ◽  
Hiroshi Sugimoto ◽  
Yu Takano ◽  
Shun Hirota ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Ligand Binding ◽  
Active Site ◽  
Redox Properties ◽  
Exogenous Ligand

scholarly journals Redox Properties of Human Medium-Chain Acyl-CoA Dehydrogenase, Modulation by Charged Active-Site Amino Acid Residues†

Biochemistry ◽  
1998 ◽  
Vol 37 (41) ◽  
pp. 14605-14612 ◽  
Author(s):  
Gina J. Mancini-Samuelson ◽  
Volker Kieweg ◽  
Kim Marie Sabaj ◽  
Sandro Ghisla ◽  
Marian T. Stankovich
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Redox Properties ◽  
Amino Acid Residues ◽  
Medium Chain ◽  
Chain Acyl

Nonlinear Vibrations in a 2-Dimensional Protein Cluster Model with Linear Bonds

2000 ◽  
Vol 214 (1) ◽  
Author(s):  
E.G. Shidlovskaya ◽  
L. Schimansky-Geier ◽  
Yu.M. Romanovsky
Keyword(s):  
Active Site ◽  
Internal Resonance ◽  
Cluster Model ◽  
Active Sites ◽  
Nonlinear Vibrations ◽  
Nonlinear Phenomena ◽  
Two Dimensional ◽  
Dimensional Model ◽  
Two Dimensional Model

A two dimensional model for the substrate inside a pocket of an active site of an enzyme is presented and investigated as a vibrational system. The parameters of the system are evaluated for α-chymotrypsin. In the case of internal resonance it is analytically and numerically shown that the energy concentrated on a certain degree of freedom might be several times larger than in the non-resonant case. Additionally, the system is driven by harmonic excitations and again energy due to nonlinear phenomena is redistributed inhomogeneously. These results may be of importance for the determination of the rates of catalytic events of substrates bound in pockets of active sites.

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.

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