Rapid Kinetic Studies and Structural Determination of a Cysteine Proteinase Mutant Imply That Residue 158 in Caricain Has a Major Effect upon the Ability of the Active Site Histidine To Protonate a Dipyridyl Probe†

Biochemistry ◽  
1996 ◽  
Vol 35 (47) ◽  
pp. 14763-14772 ◽  
Author(s):  
Nikolaos A. Katerelos ◽  
Peter W. Goodenough
Keyword(s):  
Active Site ◽  
Cysteine Proteinase ◽  
Kinetic Studies ◽  
Major Effect ◽  
Structural Determination ◽  
Active Site Histidine

Structural determination of the active site of a sweet protein A1H NMR investigation of pMNEI

FEBS Letters ◽  
1992 ◽  
Vol 310 (1) ◽  
pp. 27-30 ◽  
Author(s):  
T. Tancredi ◽  
H. Iijima ◽  
G. Saviano ◽  
P. Amodeo ◽  
P.A. Temussi
Keyword(s):  
Active Site ◽  
Structural Determination ◽  
Sweet Protein

scholarly journals Determination of active site of lysine-specific cysteine proteinase (Lys-gingipain) by use of a Porphyromonas gingivalis plasmid system

2008 ◽  
Vol 53 (6) ◽  
pp. 538-544 ◽  
Author(s):  
Yutaka Ishida ◽  
JinPing Hu ◽  
Eiko Sakai ◽  
Tomoko Kadowaki ◽  
Kenji Yamamoto ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Active Site ◽  
Cysteine Proteinase

scholarly journals Determination of the ionization state of the active-site histidine in a subtilisin-(chloromethane inhibitor) derivative by 13C-NMR

1996 ◽  
Vol 317 (1) ◽  
pp. 35-40 ◽  
Author(s):  
Timothy P. O'CONNELL ◽  
J. Paul G. MALTHOUSE
Keyword(s):  
13C Nmr ◽  
Active Site ◽  
Catalytic Mechanism ◽  
13C Nmr Spectroscopy ◽  
Imidazole Ring ◽  
Histidine Residue ◽  
Ionization State ◽  
Methylene Carbon ◽  
Active Site Histidine

Subtilisin BPN´ has been alkylated using benzyloxycarbonylglycylglycyl[1-13C]phenylalanylchloromethane. Using difference 13C-NMR spectroscopy a single signal due to the 13C-enriched α-methylene carbon of the subtilisin–(chloromethane inhibitor) derivative was detected. No evidence for the denaturation/autolysis of this derivative was obtained from pH 3.5 to 11.5. However, incubating at pH 12.75 or heating in the presence of SDS at pH 6.9 did denature this derivative. The negative titration shift of the α-methylene carbon of the denatured derivatives confirmed that the inhibitor had alkylated N-3 of the imidazole ring of the active-site histidine. The positive titration shift of 3.96 p.p.m. and the pKa of 7.04 obtained from studying the native subtilisin–(chloromethane inhibitor) derivative are assigned to oxyanion formation. We conclude that the pKa of the alkylated histidine residue in the native subtilisin–(chloromethane inhibitor) derivative must be > 12 and that subtilisin preferentially stabilizes the zwitterionic tetrahedral adduct consisting of the oxyanion and the imidazolium ion of the active-site histidine residue. We show that even before the oxyanion is formed the pKa of the active-site histidine must be much greater than that of the oxyanion in the zwitterionic tetrahedral adduct. We discuss the significance of our results for the catalytic mechanism of the serine proteinases.

Stability Indicating Liquid Chromatographic Method for the Determination of Fenofibrate and its Application to Kinetic Studies

2013 ◽  
Vol 5 (4) ◽  
pp. 1866-1874
Author(s):  
K. Srinivasa Rao ◽  
Keshar N K ◽  
N Jena ◽  
M.E.B Rao ◽  
A K Patnaik
Keyword(s):  
Degradation Products ◽  
Kinetic Studies ◽  
Pharmaceutical Formulations ◽  
Assay Method ◽  
Pharmaceutical Dosage Form ◽  
Stability Indicating ◽  
Zero Order Kinetics ◽  
Relative Standard ◽  
Liquid Chromatographic

A stability-indicating LC assay method was developed for the quantitative determination of fenofibrate (FFB) in pharmaceutical dosage form in the presence of its degradation products and kinetic determinations were evaluated in acidic, alkaline and peroxide degradation conditions. Chromatographic separation was achieved by use of Zorbax C18 column (250 × 4.0 mm, 5 μm). The mobile phase was established by mixing phosphate buffer (pH adjusted 3 with phosphoric acid) and acetonitrile (30:70 v/v). FFB degraded in acidic, alkaline and hydrogen peroxide conditions, while it was more stable in thermal and photolytic conditions. The described method was linear over a range of 1.0-500 μg/ml for determination of FFB (r= 0.9999). The precision was demonstrated by relative standard deviation (RSD) of intra-day (RSD= 0.56– 0.91) and inter-day studies (RSD= 1.47). The mean recovery was found to be 100.01%. The acid and alkaline degradations of FFB in 1M HCl and 1M NaOH solutions showed an apparent zero-order kinetics with rate constants 0.0736 and 0.0698  min−1 respectively and the peroxide degradation with 5% H2O2 demonstrated an apparent first-order kinetics with rate constant k = 0.0202 per min. The t1/2, t90   values are also determined for all the kinetic studies. The developed method was found to be simple, specific, robust, linear, precise, and accurate for the determination of FFB in pharmaceutical formulations.  

Structural Determination of Certain Novel ER Complexes

2004 ◽  
Author(s):  
Ya-Ling Wu ◽  
Geoffrey L. Greene
Keyword(s):  
Structural Determination

Phytochemical Constituents of Annona reticulata and their Cytotoxic Activity

2020 ◽  
Vol 17 (3) ◽  
pp. 206-210
Author(s):  
Ty Viet Pham ◽  
Thang Quoc Le ◽  
Anh Tuan Le ◽  
Hung Quoc Vo ◽  
Duc Viet Ho
Keyword(s):  
Cytotoxic Activity ◽  
Cell Lines ◽  
Structural Determination ◽  
Phytochemical Investigation ◽  
Ic50 Values ◽  
Phytochemical Constituents ◽  
First Time ◽  
Annona Reticulata

A phytochemical investigation of the leaves of Annona reticulata led to the isolation and structural determination of β-sitosterol (1), ent-pimara-8(14),15-dien-19-oic acid (2), ent-pimara- 8(14),15-dien-19-ol (3), quercetin (4), quercetin 3-O-α-L-arabinopyranoside (5), and a mixture of quercetin 3-O-β-D-galactopyranoside (6a) and quercetin 3-O-β-D-glucopyranoside (6b). Of these, compounds 2 and 3 were isolated from the genus Annona for the first time. Compound 3 showed strong cytotoxicity against SK-LU-1 and SW626 cell lines with IC50 values of 17.64 ± 1.07 and 19.79 ± 1.41 μg mL-1, respectively.

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