In silicoanalyses of essential interactions of iminosugars with the Hex A active site and evaluation of their pharmacological chaperone effects for Tay–Sachs disease

2017 ◽  
Vol 15 (44) ◽  
pp. 9297-9304 ◽  
Author(s):  
Atsushi Kato ◽  
Izumi Nakagome ◽  
Shinpei Nakagawa ◽  
Kyoko Kinami ◽  
Isao Adachi ◽  
...  
Keyword(s):  
Active Site ◽  
Wild Type ◽  
Pharmacological Chaperone ◽  
Tay Sachs Disease

DMDP amide restored Hex A activity in the G269S Tay–Sachs patient cells up to 43% of the wild type.

Effect of Fibrin-Like Stimulators on the Activation of Plasminogen by Tissue-Type Plasminogen Activator (t-PA) - Studies with Active Site Mutagenized Plasminogen and Plasmin Resistant t-PA

1990 ◽  
Vol 64 (01) ◽  
pp. 061-068 ◽  
Author(s):  
H R Lijnen ◽  
B Van Hoet ◽  
F De Cock ◽  
D Collen
Keyword(s):  
Active Site ◽  
Peptide Bond ◽  
Tissue Type ◽  
Wild Type ◽  
Single Chain ◽  
Plasmin Activity ◽  
Soluble Fibrin ◽  
Type Plasminogen Activator ◽  
Presence And Absence ◽  
Chain Form

SummaryThe activation of plasminogen by t-PA was measured in the presence and absence of fibrin stimulation, using natural human plasminogen (nPlg) and rPlg-Ala740, a recombinant plasminogen with the active site Ser740 mutagenaed to Ala. Recombinant wild type t-PA (rt-PA) was used as well as rt-PA -Glul275, a recombinant single chain t-PA in which the Arg of the plasmin sensitiv e Arg275- Ile276 peptide bond was substituted with Glu. Conversion of 125I-labeled single chain plasminogen to two-chain plasmin by wild-type or mutant t-PA, was quantitated by SDS gel electrophoresis and radioisotope counting of gel slices, and expressed as initial activation rates (v0 in pM s−1) per 1 μM enzyme. In the absence of fibrin stimulation, the vs for the activation of nPlg and rPlg-Ala740 with the single chain forms of both t-PAs were comparable (0.6 to 2.7 pM s−1) but were lower than with the corresponding two-chain forms (5.3 to 23 pM s−1). In the presence of 1 μM soluble fibrin monomer (desAAfibrin), the v0 for nPlg and rPlg-Ala740 by single chain rt-PA was also comparable (24 and, 33 pM s-1 respectively), whereas with 1 pM CNBr-digested fibrinogen, the vs for nPlg with single chain rt-PA was about 20-fold higher than that of rPlg-Ala740 (135 and 7.5 pM s−1 respectively). In contrast, the vs for nPlg and rPlg-Ala740 by single chain rt-PA- G1u275, two-chain rt-PA-G1u275 or two-chain rt-PA were comparable in the presence of either desAAfibrin or CNBr-digested fibrinogen.These findings confirm and establish: 1) that single chain t-PA is an active enzyme both in the presence and absence of fibrin stimulator; 2) that, in a system devoid of plasmin activity (rPlg- Ala740), the two-chain form of t-PA is about L5 times more active than the single chain form in the absence of fibrin but equipotent in the presence of desAAfibrin; and 3) that the mechanism of stimulation of plasminogen activation with single chain t-PA by CNBr-digested fibrinogen is different from that by soluble fibrin.

Discovery of natural product ovalicin sensitive type 1 methionine aminopeptidases: molecular and structural basis

2019 ◽  
Vol 476 (6) ◽  
pp. 991-1003 ◽  
Author(s):  
Vijaykumar Pillalamarri ◽  
Tarun Arya ◽  
Neshatul Haque ◽  
Sandeep Chowdary Bala ◽  
Anil Kumar Marapaka ◽  
...  
Keyword(s):  
Natural Product ◽  
Active Site ◽  
Covalent Modification ◽  
Structural Basis ◽  
Wild Type ◽  
Methionine Aminopeptidases ◽  
Synthetic Derivative ◽  
Dynamics Simulations

Abstract Natural product ovalicin and its synthetic derivative TNP-470 have been extensively studied for their antiangiogenic property, and the later reached phase 3 clinical trials. They covalently modify the conserved histidine in Type 2 methionine aminopeptidases (MetAPs) at nanomolar concentrations. Even though a similar mechanism is possible in Type 1 human MetAP, it is inhibited only at millimolar concentration. In this study, we have discovered two Type 1 wild-type MetAPs (Streptococcus pneumoniae and Enterococcus faecalis) that are inhibited at low micromolar to nanomolar concentrations and established the molecular mechanism. F309 in the active site of Type 1 human MetAP (HsMetAP1b) seems to be the key to the resistance, while newly identified ovalicin sensitive Type 1 MetAPs have a methionine or isoleucine at this position. Type 2 human MetAP (HsMetAP2) also has isoleucine (I338) in the analogous position. Ovalicin inhibited F309M and F309I mutants of human MetAP1b at low micromolar concentration. Molecular dynamics simulations suggest that ovalicin is not stably placed in the active site of wild-type MetAP1b before the covalent modification. In the case of F309M mutant and human Type 2 MetAP, molecule spends more time in the active site providing time for covalent modification.

scholarly journals Active-site serine mutants of the Streptomyces albus G β-lactamase

1991 ◽  
Vol 277 (3) ◽  
pp. 647-652 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Ph Values ◽  
Streptomyces Albus ◽  
Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

Effects of viscosity and osmotic stress on the reaction of human butyrylcholinesterase with cresyl saligenin phosphate, a toxicant related to aerotoxic syndrome: kinetic and molecular dynamics studies

2013 ◽  
Vol 454 (3) ◽  
pp. 387-399 ◽  
Author(s):  
Patrick Masson ◽  
Sofya Lushchekina ◽  
Lawrence M. Schopfer ◽  
Oksana Lockridge
Keyword(s):  
Osmotic Stress ◽  
Osmotic Pressure ◽  
Active Site ◽  
Md Simulations ◽  
Catalytic Triad ◽  
Wild Type ◽  
Irreversible Inhibitor ◽  
Enzyme Active Site ◽  
Diffusion Controlled ◽  
Peripheral Site

CSP (cresyl saligenin phosphate) is an irreversible inhibitor of human BChE (butyrylcholinesterase) that has been involved in the aerotoxic syndrome. Inhibition under pseudo-first-order conditions is biphasic, reflecting a slow equilibrium between two enzyme states E and E′. The elementary constants for CSP inhibition of wild-type BChE and D70G mutant were determined by studying the dependence of inhibition kinetics on viscosity and osmotic pressure. Glycerol and sucrose were used as viscosogens. Phosphorylation by CSP is sensitive to viscosity and is thus strongly diffusion-controlled (kon≈108 M−1·min−1). Bimolecular rate constants (ki) are about equal to kon values, making CSP one of the fastest inhibitors of BChE. Sucrose caused osmotic stress because it is excluded from the active-site gorge. This depleted the active-site gorge of water. Osmotic activation volumes, determined from the dependence of ki on osmotic pressure, showed that water in the gorge of the D70G mutant is more easily depleted than that in wild-type BChE. This demonstrates the importance of the peripheral site residue Asp70 in controlling the active-site gorge hydration. MD simulations provided new evidence for differences in the motion of water within the gorge of wild-type and D70G enzymes. The effect of viscosogens/osmolytes provided information on the slow equilibrium E⇌E′, indicating that alteration in hydration of a key catalytic residue shifts the equilibrium towards E′. MD simulations showed that glycerol molecules that substitute for water molecules in the enzyme active-site gorge induce a conformational change in the catalytic triad residue His438, leading to the less reactive form E′.

Enzyme activity enhancement of chondroitinase ABC I from Proteus vulgaris by site-directed mutagenesis

RSC Advances ◽  
2015 ◽  
Vol 5 (93) ◽  
pp. 76040-76047 ◽  
Author(s):  
Zhenya Chen ◽  
Ye Li ◽  
Yue Feng ◽  
Liang Chen ◽  
Qipeng Yuan
Keyword(s):  
Active Site ◽  
Simulation Analysis ◽  
Docking Simulation ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Proteus Vulgaris ◽  
Molecular Docking Simulation ◽  
Activity Enhancement ◽  
First Time

Arg660 was found as a new active site and Asn795Ala and Trp818Ala mutants showed higher activities than the wild type based on molecular docking simulation analysis for the first time.

scholarly journals Glutamic dehydrogenase in revertants of am mutants in neurospora

1966 ◽  
Vol 7 (1) ◽  
pp. 18-31 ◽  
Author(s):  
David R. Stadler
Keyword(s):  
Genetic Analysis ◽  
Active Site ◽  
Ultra Violet ◽  
Wild Type ◽  
Glutamic Dehydrogenase

Five allelic mutants of Neurospora which lack glutamic dehydrogenase (am mutants) were induced to revert with ultra-violet. The glutamic dehydrogenase produced by the revertants was compared to that of wild-type. Several distinct classes of revertants could be distinguished by these tests. However, genetic analysis showed that all the reversions resulted from events at or near the site of the original am mutation. The spectrum of reversion types depended on the nature of the am mutant employed. One mutant, which produces an am protein believed to be altered in its active site, yielded revertants which were all indistinguishable from wild-type. Another mutant, which produces a protein with a functional active site but altered folding properties, gave at least six classes of revertants which were different from wild-type.

scholarly journals Uncovering the Role of Key Active Site Side Chains in Catalysis: An Extended Brønsted Relationship for Substrate Deprotonation Catalysed by Wild-Type and Variants of Triosephosphate Isomerase

2019 ◽  
Author(s):  
Yashraj S. Kulkarni ◽  
Tina L. Amyes ◽  
John Richard ◽  
Shina Caroline Lynn Kamerlin
Keyword(s):  
Active Site ◽  
Triosephosphate Isomerase ◽  
Valence Bond ◽  
Side Chains ◽  
Wild Type ◽  
Empirical Valence Bond

Manuscript and supporting information outlining an analysis of an extended Brønsted relationship obtained from empirical valence bond simulations of substrate deprotonation catalyzed by wild-type and mutant variants of triosephosphate isomerase.

scholarly journals Residual Helicity at the Active Site of the Histidine Phosphocarrier, HPr, Modulates Binding Affinity to Its Natural Partners

2021 ◽  
Vol 22 (19) ◽  
pp. 10805
Author(s):  
José L. Neira ◽  
David Ortega-Alarcón ◽  
Bruno Rizzuti ◽  
Martina Palomino-Schätzlein ◽  
Adrián Velázquez-Campoy ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Active Site ◽  
Antibacterial Properties ◽  
Helical Structure ◽  
Titration Calorimetry ◽  
Its Sequence ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Residual Structure ◽  
Α Helix

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first α-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr9–30), involving the first α-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr9–30 is mainly disordered. We attempted to improve the affinity of HPr9–30 to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr9–30. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. Therefore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function.

scholarly journals Sensitivity of the C-Terminal Nuclease Domain of Kaposi's Sarcoma-Associated Herpesvirus ORF29 to Two Classes of Active-Site Ligands

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
Jennifer T. Miller ◽  
Haiyan Zhao ◽  
Takashi Masaoka ◽  
Brittany Varnado ◽  
Elena M. Cornejo Castro ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Active Site ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Nuclease Activity ◽  
Divalent Metal ◽  
Wild Type ◽  
Nuclease Domain ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma, belongs to theHerpesviridaefamily, whose members employ a multicomponent terminase to resolve nonparametric viral DNA into genome-length units prior to their packaging. Homology modeling of the ORF29 C-terminal nuclease domain (pORF29C) and bacteriophage Sf6 gp2 have suggested an active site clustered with four acidic residues, D476, E550, D661, and D662, that collectively sequester the catalytic divalent metal (Mn2+) and also provided important insight into a potential inhibitor binding mode. Using this model, we have expressed, purified, and characterized the wild-type pORF29C and variants with substitutions at the proposed active-site residues. Differential scanning calorimetry demonstrated divalent metal-induced stabilization of wild-type (WT) and D661A pORF29C, consistent with which these two enzymes exhibited Mn2+-dependent nuclease activity, although the latter mutant was significantly impaired. Thermal stability of WT and D661A pORF29C was also enhanced by binding of an α-hydroxytropolone (α-HT) inhibitor shown to replace divalent metal at the active site. For the remaining mutants, thermal stability was unaffected by divalent metal or α-HT binding, supporting their role in catalysis. pORF29C nuclease activity was also inhibited by two classes of small molecules reported to inhibit HIV RNase H and integrase, both of which belong to the superfamily of nucleotidyltransferases. Finally, α-HT inhibition of KSHV replication suggests ORF29 nuclease function as an antiviral target that could be combined with latency-activating compounds as a shock-and-kill antiviral strategy.

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