scholarly journals Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae

PLoS ONE ◽  
2016 ◽  
Vol 11 (6) ◽  
pp. e0156892 ◽  
Author(s):  
Bastian Daniel ◽  
Silvia Wallner ◽  
Barbara Steiner ◽  
Gustav Oberdorfer ◽  
Prashant Kumar ◽  
...  
Keyword(s):  
Active Site ◽  
Plant Family ◽  
Site Specific ◽  
Berberine Bridge Enzyme

Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine

1992 ◽  
Vol 12 (9) ◽  
pp. 3757-3765
Author(s):  
J W Chen ◽  
B R Evans ◽  
S H Yang ◽  
H Araki ◽  
Y Oshima ◽  
...  
Keyword(s):  
Active Site ◽  
Dna Cleavage ◽  
Substrate Binding ◽  
Point Mutations ◽  
Transfer Reactions ◽  
Strand Transfer ◽  
Site Specific ◽  
Arginine Residues ◽  
Active Site Tyrosine ◽  
Half Site

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.

BIS-ANS Binding To Human Thrombins

1981 ◽  
Author(s):  
G Metz ◽  
L J Berliner
Keyword(s):  
Active Site ◽  
Unique Feature ◽  
Enhanced Fluorescence ◽  
Ans Binding ◽  
Site Specific ◽  
Catalytic Center ◽  
Strong Binding

Bis-anilinonaphthalenesulfonate(bis-ANS) binds extremely strongly to the human thrombins accompanied by a much enhanced fluorescence for the dye. One unique feature of bis-ANS binding is an enhancement of TAME hydrolysis by α-thrombin to approximately 175% times the rate for native α-thrombin at pH 8.1 (0 M NaCl). Non-coagulant γ-thrombin is distinguished from highly coagulant α-thrombin by enhancing bis-ANS fluorescence ∼2 times more than that by the latter form. The very strong binding of this dye Kdiss<10-8 M, pH 6.5, 0.05 M Phosphate, 0.75 M NaCl) as welras its apparent binding loci situated near, but not obstructing, the catalytic center displays the powerfully useful properties of this probe, extremely sensitive to thrombin environment while not inhibiting its action. This is to be compared with the active site specific fluorophore inhibitor DAPA which prevents binding of other ligands to the thrombin active site.

scholarly journals Oxidative inactivation of plasmin and other serine proteases by copper and ascorbate

Blood ◽  
1993 ◽  
Vol 82 (5) ◽  
pp. 1522-1531 ◽  
Author(s):  
SE Lind ◽  
JR McDonagh ◽  
CJ Smith
Keyword(s):  
Plasminogen Activator ◽  
Active Site ◽  
Serine Proteases ◽  
Plasminogen Activators ◽  
Single Chain ◽  
Site Specific ◽  
Oxidative Inactivation ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Specific Oxidation

Abstract Fibrin thrombi form at sites of injury, where leukocytes release a variety of oxidants. To determine whether oxidants might affect proteins of the fibrinolytic system, we examined the effects of various oxidants on plasmin. Plasmin was not inhibited by micromolar concentrations of hypochlorous acid, chloramine T, or H2O2. Neither Fe nor Cu affected plasmin alone or in the presence of H2O2. However, incubation of plasmin with 5 mumol/L Cu(I or II) in the presence of the reducing agent ascorbic acid resulted in a loss of its hydrolytic activity towards proteins as well as towards small synthetic substrates. The addition of EDTA, but not mannitol, prevented its inactivation. Inactivation was prevented by the addition of catalase and accelerated by hydrogen peroxide. Preincubation of plasmin with the competitive inhibitor alpha-N-acetyl-L-lysine methyl ester prevented inactivation by Cu(II) and ascorbate. These results together suggest site-specific oxidation of plasmin's active site. Treatment of the plasminogen activators tissue plasminogen activator and two-chain urokinase-type plasminogen activator, as well as trypsin, neutrophil elastase, and thrombin with Cu(II) and ascorbate resulted in a loss of their amidolytic and proteolytic activity, indicating the general susceptibility of serine proteases to this type of oxidation. Oxidation of the zymogens Glu-plasminogen and single-chain urokinase-type plasminogen activator by Cu(II) and ascorbate resulted in the failure of these molecules to generate active enzymes when treated with plasminogen activators or plasmin, respectively. The active site His residue may be the target of oxidative inactivation, as evidenced by the partial protection afforded plasmin by the addition of Zn(II), histidine, or the platinum derivative, platinum(II) (2,2′:6′,2″- terpyridine) chloride. Because platelets contain micromolar concentrations of Cu and leukocytes are rich in ascorbate, Cu-dependent site-specific oxidation might play a role in modulating proteolytic events and the life span of thrombi formed at sites of tissue injury.

scholarly journals Site-Specific, Intramolecular Cross-Linking of Pin1 Active Site Residues by the Lipid Electrophile 4-Oxo-2-nonenal

2015 ◽  
Vol 28 (4) ◽  
pp. 817-827 ◽  
Author(s):  
Christopher D. Aluise ◽  
Jeannie M. Camarillo ◽  
Yuki Shimozu ◽  
James J. Galligan ◽  
Kristie L. Rose ◽  
...  
Keyword(s):  
Active Site ◽  
Cross Linking ◽  
Active Site Residues ◽  
Site Specific

Postproline cleaving enzyme: identification as serine protease using active site specific inhibitors

Biochemistry ◽  
1977 ◽  
Vol 16 (13) ◽  
pp. 2942-2948 ◽  
Author(s):  
Tadashi Yoshimoto ◽  
Ronald C. Orlowski ◽  
Roderich Walter
Keyword(s):  
Serine Protease ◽  
Active Site ◽  
Site Specific ◽  
Specific Inhibitors

scholarly journals Engineering a light-controlled F1ATPase using structure-based protein design

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2286 ◽  
Author(s):  
Daniel Hoersch
Keyword(s):  
Protein Design ◽  
Active Site ◽  
Atp Hydrolysis ◽  
Site Specific ◽  
Dynamic Constraint ◽  
End Distance ◽  
A Site ◽  
Hydrolysis Activity

The F1sub-complex of ATP synthase is a biological nanomotor that converts the free energy of ATP hydrolysis into mechanical work with an astonishing efficiency of up to 100% (Kinosita et al., 2000). To probe the principal mechanics of the machine, I re-engineered the active site ofE.coliF1ATPase with a structure-based protein design approach: by incorporation of a site-specific, photoswitchable crosslinker, whose end-to-end distance can be modulated by illumination with light of two different wavelengths, a dynamic constraint was imposed on the inter-atomic distances of the α and β subunits. Crosslinking reduced the ATP hydrolysis activity of four designs tested in vitro and in one case created a synthetic ATPase whose activity can be reversibly modulated by subsequent illumination with near UV and blue light. The work is a first step into the direction of the long-term goal to design nanoscaled machines based on biological parts that can be precisely controlled by light.

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