scholarly journals Intra-dimer cooperativity between the active site cysteines during the oxidation of peroxiredoxin 2

2020 ◽  
Author(s):  
Alexander V Peskin ◽  
Flávia C Meotti ◽  
Luiz F de Souza ◽  
Robert F Anderson ◽  
Christine C Winterbourn ◽  
...  
Keyword(s):  
Active Site ◽  
Disulfide Bonds ◽  
Flow Analysis ◽  
Kinetic Studies ◽  
Tryptophan Fluorescence ◽  
Negative Cooperativity ◽  
Sulfenic Acid ◽  
Peroxiredoxin 2 ◽  
Active Site Cysteine ◽  
Sds Page

ABSTRACTPeroxiredoxin 2 (Prdx2) and other typical 2-Cys Prdxs function as homodimers in which hydrogen peroxide oxidizes each active site cysteine to a sulfenic acid which then condenses with the resolving cysteine on the alternate chain. Previous kinetic studies have considered both sites as equally reactive. Here we have studied Prdx2 using a combination of non-reducing SDS-PAGE to separate reduced monomers and dimers with one and two disulfide bonds, and stopped flow analysis of tryptophan fluorescence, to investigate whether there is cooperativity between the sites. We have observed positive cooperativity when H2O2 is added as a bolus and oxidation of the second site occurs while the first site is present as a sulfenic acid. Modelling of this reaction showed that the second site reacts 2.2 ± 0.1 times faster. In contrast, when H2O2 was generated slowly and the first active site condensed to a disulfide before the second site reacted, no cooperativity was evident. Conversion of the sulfenic acid to the disulfide showed negative cooperativity, with modelling of the exponential rise in tryptophan fluorescence yielding a rate constant of 0.75 ± 0.08 s-1 when the alternate active site was present as a sulfenic acid and 2.29 ± 0.08-fold lower when it was a disulfide. No difference in the rate of hyperoxidation at the two sites was detected. Our findings imply that oxidation of one active site affects the conformation of the second site and influences which intermediate forms of the protein are favored under different cellular conditions.

scholarly journals Transient-kinetic studies of the adenosine triphosphatase activity of scallop heavy meromyosin

1988 ◽  
Vol 251 (2) ◽  
pp. 515-526 ◽  
Author(s):  
A P Jackson ◽  
C R Bagshaw
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Kinetic Studies ◽  
Tryptophan Fluorescence ◽  
Adductor Muscle ◽  
Reverse Direction ◽  
Heavy Meromyosin ◽  
Thin Filaments ◽  
Adp Release ◽  
Atp Binding And Hydrolysis

Fluorescence stopped-flow experiments were performed to elucidate the elementary steps of the ATPase mechanism of scallop heavy meromyosin in the presence and in the absence of Ca2+. ATP binding and hydrolysis, as monitored by the change in tryptophan fluorescence, appear to be Ca2+-insensitive, whereas both Pi release and ADP release are markedly suppressed in the absence of Ca2+. Rate constants for Pi release are 0.2 s-1 and 0.002 s-1 and for ADP release are 6 s-1 and 0.01 s-1 in the presence and in the absence of Ca2+ respectively. Ca2+ binding to the specific site of the regulatory domain is rapid and its release occurs at 25 s-1, consistent with the time scale of a twitch of the striated adductor muscle. Nucleotide binding is a multi-step process requiring a minimum of three states. In such a model Ca2+ controls the rate of conformational changes at the active site in both the forward and the reverse direction, leading to a large dependence of the rate of nucleotide release, but a lesser effect on the overall equilibrium position. The kinetic trapping of nucleotides and Pi at the active site, in the absence of Ca2+, appears to be a fundamental step in suppressing the interaction of the myosin head with the thin filaments in relaxed molluscan muscle.

scholarly journals Identification of the active site cysteine and of the disulfide bonds in the N-terminal part of the molecule of bovine spleen cathepsin B

FEBS Letters ◽  
1982 ◽  
Vol 142 (1) ◽  
pp. 23-26 ◽  
Author(s):  
Jan Pohl ◽  
Miroslav Baudyš ◽  
Vladimír Tomášek ◽  
Vladimír Kostka
Keyword(s):  
Active Site ◽  
Cathepsin B ◽  
Disulfide Bonds ◽  
Terminal Part ◽  
Active Site Cysteine

scholarly journals Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nur Suhanawati Ashaari ◽  
Mohd Hairul Ab. Rahim ◽  
Suriana Sabri ◽  
Kok Song Lai ◽  
Adelene Ai-Lian Song ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Homology Model ◽  
Biochemical Properties ◽  
Terpene Synthase ◽  
Monoterpene Synthase ◽  
Terminal Domain ◽  
Catalytic Active Site ◽  
Plectranthus Amboinicus

AbstractLinalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis–Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10–3 µM−1 s−1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.

Alkylation of Escherichia coli Thioredoxin by S-(2-Chloroethyl)glutathione and Identification of the Adduct on the Active Site Cysteine-32 by Mass Spectrometry

1995 ◽  
Vol 8 (7) ◽  
pp. 934-941 ◽  
Author(s):  
John C. L. Erve ◽  
Elisabeth Barofsky ◽  
Douglas F. Barofsky ◽  
Max L. Deinzer ◽  
Donald J. Reed
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Active Site ◽  
Active Site Cysteine
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