scholarly journals A new chromophoric substrate for penicillopepsin and other fungal aspartic proteinases

1982 ◽  
Vol 203 (3) ◽  
pp. 603-610 ◽  
Author(s):  
Theo Hofmann ◽  
Robert S. Hodges
Keyword(s):  
Catalytic Efficiency ◽  
Aspartic Proteinase ◽  
Spectrophotometric Assay ◽  
Water Soluble ◽  
Burst Release ◽  
Penicillium Roqueforti ◽  
Aspartic Proteinases ◽  
Catalytic Rate Constant ◽  
Human Renin ◽  
Hydrolysis Of

The hexapeptide N-α-acetylalanylalanyl-lysyl-p- nitrophenylalanylalanylalanylamide has been synthesized and was found to be a good substrate for fungal aspartic proteinases that possess trypsinogen-activating activity, namely penicillopepsin, Rhizopus aspartic proteinase, Endothia aspartic proteinase and the aspartic proteinases from Aspergillus oryzae and Penicillium roqueforti. The peptide is rapidly cleaved between the lysine and p-nitrophenylalanine residues. Calf chymosin and human renin cleave the same bond, but only very slowly. The cleavage is accompanied by an absorbance decrease with a maximum at 296nm (Δε —1800m−1·cm−1). Pig pepsin and the aspartic proteinases from two Rhizomucor species cleave the peptide slowly on the carboxy side of p-nitrophenylalanine. For the five enzymes that hydrolysed the peptide rapidly, Km values range from 0.16 to 0.42mm and kcat. from 6 to 46.6s−1 at pH 4.5 and 25°C. A comparison of the kinetic parameters of the hexapeptide with those of the dipeptide N-α-acetyllysyl-p-nitrophenylalanylamide obtained with penicillopepsin shows that at pH 6.0 the catalytic rate constant kcat. is over 5000-fold greater for the hexapeptide, whereas the Km values are essentially the same, showing that the catalytic efficiency is strongly dependent on secondary binding. The new substrate with a p-nitrophenylalanine residue in the P′1 position has advantages over previously used substrates for aspartic proteinases in that it offers a more sensitive spectrophotometric assay that is independent of pH up to 5.5 and can readily be used up to pH 7.0. The presence of lysine makes it very water-soluble. Stopped-flow spectrophotometric experiments with penicillopepsin gave clear evidence that the hydrolysis of the substrate by penicillopepsin is not accompanied by a ‘burst’ release of p-nitrophenylalanylalanylalanylamide.

scholarly journals A systematic series of synthetic chromophoric substrates for aspartic proteinases

1986 ◽  
Vol 237 (3) ◽  
pp. 899-906 ◽  
Author(s):  
B M Dunn ◽  
M Jimenez ◽  
B F Parten ◽  
M J Valler ◽  
C E Rolph ◽  
...  
Keyword(s):  
Structure Function ◽  
Peptide Bond ◽  
Water Soluble ◽  
Animal Origin ◽  
Aspartic Proteinases ◽  
Peptide Substrates ◽  
Representative Selection ◽  
Micro Organisms ◽  
Hydrolysis Of ◽  
Selection Of

The hydrolysis of the chromogenic peptide Pro-Thr-Glu-Phe-Phe(4-NO2)-Arg-Leu at the Phe-Phe(4-NO2) bond by nine aspartic proteinases of animal origin and seven enzymes from micro-organisms is described [Phe(4-NO2) is p-nitro-L-phenylalanine]. A further series of six peptides was synthesized in which the residue in the P3 position was systematically varied from hydrophobic to hydrophilic. The Phe-Phe(4-NO2) bond was established as the only peptide bond cleaved, and kinetic constants were obtained for the hydrolysis of these peptide substrates by a representative selection of aspartic proteinases of animal and microbial origin. The value of these water-soluble substrates for structure-function investigations is discussed.

scholarly journals The selectivity of statine-based inhibitors against various human aspartic proteinases

1990 ◽  
Vol 265 (3) ◽  
pp. 871-878 ◽  
Author(s):  
R A Jupp ◽  
B M Dunn ◽  
J W Jacobs ◽  
G Vlasuk ◽  
K E Arcuri ◽  
...  
Keyword(s):  
Cathepsin D ◽  
Aspartic Proteinase ◽  
The Other ◽  
Aspartic Proteinases ◽  
Cathepsin E ◽  
Human Renin ◽  
Endothia Parasitica ◽  
Selection Of

The interactions of five human enzymes (renin, pepsin, gastricsin, cathepsin D and cathepsin E) and the aspartic proteinase from Endothia parasitica with several series of synthetic inhibitors were examined. All of the inhibitors contained the dipeptide analogue statine or its phenylalanine or cyclohexylalanine homologues in the P1-P1′ positions. The residues occupying the peripheral sub-sites (P4 to P3′) were varied systematically and inhibitory constants were determined for the interactions with each of the proteinases. Inhibitors were elucidated that specifically inhibited human renin and did not affect any of the other human enzymes or the fungal proteinase. With suitable selection of residues to occupy individual sub-sites, effective inhibitors of specific human aspartic proteinases may now be designed.

Barley arabinoxylan arabinofuranohydrolases: purification, characterization and determination of primary structures from cDNA clones

2001 ◽  
Vol 356 (1) ◽  
pp. 181-189 ◽  
Author(s):  
Robert C. LEE ◽  
Rachel A. BURTON ◽  
Maria HRMOVA ◽  
Geoffrey B. FINCHER
Keyword(s):  
Catalytic Efficiency ◽  
Exchange Chromatography ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Hordeum Vulgare L ◽  
Ph Optimum ◽  
Catalytic Rate Constant ◽  
Arabinoxylan Arabinofuranohydrolase ◽  
Mature Enzyme ◽  
Hydrolysis Of

A family 51 arabinoxylan arabinofuranohydrolase, designated AXAH-I, has been purified from extracts of 7-day-old barley (Hordeum vulgare L.) seedlings by fractional precipitation with (NH4)2SO4 and ion-exchange chromatography. The enzyme has an apparent molecular mass of 65kDa and releases l-arabinose from cereal cell wall arabinoxylans with a pH optimum of 4.3, a catalytic rate constant (kcat) of 6.9s−1 and a catalytic efficiency factor (kcat/Km) of 0.76 (ml·s−1·mg−1). Whereas the hydrolysis of α-l-arabinofuranosyl residues linked to C(O)3 of backbone (1 → 4)-β-xylosyl residues proceeds at the fastest rate, α-l-arabinofuranosyl residues on doubly substituted xylosyl residues are also hydrolysed, at lower rates. A near full-length cDNA encoding barley AXAH-I indicates that the mature enzyme consists of 626 amino acid residues and has a calculated pI of 4.8. A second cDNA, which is 81% identical with that encoding AXAH-I, encodes another barley AXAH, which has been designated AXAH-II. The barley AXAHs are likely to have key roles in wall metabolism in cereals and other members of the Poaceae. Thus the enzymes could participate in the modification of the fine structure of arabinoxylan during wall deposition, maturation or expansion, or in wall turnover and the hydrolysis of arabinoxylans in germinated grain.

Formaldehyde as an Active Site Label of α-Chymotrypsin

1972 ◽  
Vol 50 (10) ◽  
pp. 1114-1121 ◽  
Author(s):  
Charles J. Martin ◽  
Narendra B. Oza ◽  
Mario A. Marini
Keyword(s):  
Active Site ◽  
Competitive Inhibition ◽  
Catalytic Efficiency ◽  
Formaldehyde Concentration ◽  
Binding Studies ◽  
Photometric Analysis ◽  
Limit Values ◽  
Catalytic Rate Constant ◽  
Catalytic Rate ◽  
Hydrolysis Of

The effect of formaldehyde concentration on the steady state parameters of the chymotrypsin-catalyzed hydrolysis of specific ester substrates has been investigated. At the limit values which are attained in about 3 M formaldehyde, the catalytic rate constant is decreased eightfold and [Formula: see text] increased ca. 2.5-foid. The reduction in activity does not conform to competitive, noncompetitive, uncompetitive, or partially competitive inhibition. The effect of increasing formaldehyde concentration is, however, in accord with an equimolar equilibrium reaction between chymotrypsin and formaldehyde with Kassoc of 5 M−1. Spectro-photometric analysis of the same reaction (J. Biol. Chem. 242, 5736 (1967)) and binding studies with 14C-elabeled formaldehyde (Biochim. Biophys. Acta, 242, 320 (1971)) have shown that both histidine residues react with formaldehyde.From a study of the imidazole group catalyzed hydrolysis of p-nitrophenyl acetate, the effect of formaldehyde on such processes is qualitatively similar to that observed for reactions catalyzed by chymotrypsin. The Kassoc values, however, are higher: 36 M−1 for imidazole and 25 M−1 for acetylhistidine.On the basis of these and other results, it is concluded that formaldehyde reacts with the active site of chymotrypsin to form a modified enzyme with decreased catalytic efficiency. The causative event is most probably due to the formation of an N-hydroxymethyl derivative of the His-57 residue.

scholarly journals Construction, expression and characterization of a chimaeric mammalian-plant aspartic proteinase

2003 ◽  
Vol 372 (3) ◽  
pp. 671-678 ◽  
Author(s):  
Kenneth G. PAYIE ◽  
Takuji TANAKA ◽  
Susannah GAL ◽  
Rickey Y. YADA
Keyword(s):  
Temperature Stability ◽  
Catalytic Efficiency ◽  
Aspartic Proteinase ◽  
Enzymic Activity ◽  
Scanning Calorimetry ◽  
Aspartic Proteinases ◽  
Specific Sequence ◽  
Enzyme Families ◽  
First Time

Aspartic proteinases are a well-characterized class of proteinases. In plants, all nascent aspartic proteinases possess a 100-amino-acid, plant-specific sequence (PSS) within their C-terminal lobe, presumed to possess a targeting role in vivo. In this study, the PSS domain from the Arabidopsis thaliana aspartic proteinase was inserted into porcine pepsinogen at the identical location found in nascent plant aspartic proteinases, to create a chimaeric mammalian–plant enzyme. Based on enzymic activity, this chimaeric enzyme demonstrated increases in pH stability above 6 and temperature stability above 60 °C compared with commercial pepsin. Differential scanning calorimetry of the chimaeric enzyme illustrated an approx. 2 °C increase in denaturation temperature (Tm), with increases in co-operativity and similar enthalpy values. Kinetic analysis indicated an increase in Km and decreased kcat compared with pepsin, but with a catalytic efficiency similar to the monomeric plant aspartic proteinase from wheat. Using oxidized insulin B-chain, the chimaeric enzyme demonstrated more restricted substrate specificity in comparison with commercial pepsin. This study highlights the use of a chimaeric enzyme strategy in order to characterize unique protein domains within enzyme families, and, for the first time, a putative structure–function role for the PSS as it pertains to plant aspartic proteinases.

Water Soluble Silicon Quantum Dots Grafted with Amoxicillin as a Drug Delivery System

2020 ◽  
Vol 20 (8) ◽  
pp. 4624-4628 ◽  
Author(s):  
Dowoo Ki ◽  
Honglae Sohn
Keyword(s):  
Drug Delivery ◽  
Quantum Dots ◽  
Controlled Release ◽  
Ultra Violet ◽  
Water Soluble ◽  
Burst Release ◽  
Potential Candidate ◽  
Silicon Quantum Dots ◽  
Hydrolysis Of ◽  
Soluble Silicon

Water soluble silicon quantum dots (Si QDs) are prepared and used for the measurement of the efficiency for the controlled release drug delivery. Amoxicillin, antibiotics, is covalently grafted with Si QDs through a surface derivatization reaction. Si QDs are embedded in hydrogel polymer. The release of amoxicillin has been measured by using Ultra violet-visible (UV-vis) absorption spectrometer. Amoxicillin-embedded hydrogels exhibit a burst release for 1 h, however covalently loaded Amoxi-Si QDs hydrogel composite exhibited very slow release. Absorption analysis reveals that the Si QDs exhibits a great potential candidate for controlled release of drug. The controlled drugrelease profiles depend on the hydrolysis of amoxicillin from the surface of Si QDs.

scholarly journals Optimizing Chitin Depolymerization by Lysozyme to Long-Chain Oligosaccharides

Marine Drugs ◽  
2021 ◽  
Vol 19 (6) ◽  
pp. 320
Author(s):  
Arnaud Masselin ◽  
Antoine Rousseau ◽  
Stéphanie Pradeau ◽  
Laure Fort ◽  
Rodolphe Gueret ◽  
...  
Keyword(s):  
Time Course ◽  
Water Soluble ◽  
Organic Fertilizers ◽  
Symbiotic Associations ◽  
Egg White Lysozyme ◽  
Hen Egg White ◽  
Ecological Transition ◽  
Optimized Conditions ◽  
Hydrolysis Of ◽  
Long Chain

Chitin oligosaccharides (COs) hold high promise as organic fertilizers in the ongoing agro-ecological transition. Short- and long-chain COs can contribute to the establishment of symbiotic associations between plants and microorganisms, facilitating the uptake of soil nutrients by host plants. Long-chain COs trigger plant innate immunity. A fine investigation of these different signaling pathways requires improving the access to high-purity COs. Here, we used the response surface methodology to optimize the production of COs by enzymatic hydrolysis of water-soluble chitin (WSC) with hen egg-white lysozyme. The influence of WSC concentration, its acetylation degree, and the reaction time course were modelled using a Box–Behnken design. Under optimized conditions, water-soluble COs up to the nonasaccharide were formed in 51% yield and purified to homogeneity. This straightforward approach opens new avenues to determine the complex roles of COs in plants.

THE WATER-SOLUBLE POLYSACCHARIDES OF DERMATOPHYTES: IV. GALACTOMANNANS I FROM TRICHOPHYTON GRANULOSUM, TRICHOPHYTON INTERDIGITALE, MICROSPORUM QUINCKEANUM, TRICHOPHYTON RUBRUM, AND TRICHOPHYTON SCHÖNLEINII

1965 ◽  
Vol 43 (1) ◽  
pp. 30-39 ◽  
Author(s):  
C. T. Bishop ◽  
M. B. Perry ◽  
F. Blank ◽  
F. P. Cooper
Keyword(s):  
Aqueous Solutions ◽  
Copper Complexes ◽  
Trichophyton Rubrum ◽  
Periodate Oxidation ◽  
Structural Features ◽  
Water Soluble ◽  
Major Constituent ◽  
Branch Points ◽  
Terminal Units ◽  
Hydrolysis Of

A group of polysaccharides, called galactomannans I, were precipitated as their insoluble copper complexes from aqueous solutions of the crude polysaccharides obtained from each of the organisms designated in the title. The five galactomannans I were homogeneous under conditions of electrophoresis and ultracentrifugation and had high positive specific rotations. The major constituent monosaccharide was D-mannose; amounts of D-galactose ranged from nil for the polysaccharide from T. rubrum to 13% for that from T. schönleinii. Methylation and hydrolysis of the five galactomannans I yielded varying amounts of the following: 2,3,5,6-tetra-O-methyl-D-galactose (not present in the products from T. rubrum), 2,3,4,6-tetra-O-methyl-D-mannose, 2,3,4-tri-O-methyl-D-mannose, 2,4,6-tri-O-methyl-D-mannose, 3,4-di-O-methyl-D-mannose, and 3,5-di-O-methyl-D-mannose. Periodate oxidation results agreed with the methylation studies. The gross structural features of each galactomannan I appear to be the same, namely, a basic chain of 1 → 6 linked α-D-mannopyranose units for approximately every 22 of which there is a 1 → 3 linked α-D-mannopyranose residue. Branch points occur along the 1 → 6 linked chain at the C2 positions of the D-mannopyranose units and once in every 45 units at the C2 position of a 1 → 6 linked D-mannofuranose residue. The D-galactose in the polysaccharides is present exclusively as non-reducing terminal furanose units; non-reducing terminal units of D-mannopyranose are also present. The variations in the identities and relative amounts of the non-reducing terminal units were the only apparent differences in the gross structural features within this group of polysaccharides.

scholarly journals Cover Feature: Distance between Metal Centres Affects Catalytic Efficiency of Dinuclear CoIII Complexes in the Hydrolysis of a Phosphate Diester (Eur. J. Org. Chem. 39/2018)

2018 ◽  
Vol 2018 (39) ◽  
pp. 5335-5335
Author(s):  
Eva Szusanna Bencze ◽  
Cristiano Zonta ◽  
Fabrizio Mancin ◽  
Leonard J. Prins ◽  
Paolo Scrimin
Keyword(s):  
Catalytic Efficiency ◽  
Phosphate Diester ◽  
Hydrolysis Of
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