Kinetic Characterization of the Peptidase Activity ofEscherichia coliLon Reveals the Mechanistic Similarities in ATP-Dependent Hydrolysis of Peptide and Protein Substrates

Biochemistry ◽  
2002 ◽  
Vol 41 (30) ◽  
pp. 9418-9425 ◽  
Author(s):  
Jennifer Thomas-Wohlever ◽  
Irene Lee
Keyword(s):  
Peptidase Activity ◽  
Kinetic Characterization ◽  
Protein Substrates ◽  
Hydrolysis Of

Characterization of peptidases of adult Trichuris muris

Parasitology ◽  
1994 ◽  
Vol 109 (5) ◽  
pp. 623-630 ◽  
Author(s):  
L. J. Drake ◽  
A. E. Bianco ◽  
D. A. P. Bundy ◽  
F. Ashall
Keyword(s):  
Mammalian Cells ◽  
Culture Medium ◽  
Matrix Protein ◽  
Sodium Dodecyl ◽  
Time Dependent ◽  
Extracellular Matrix Protein ◽  
Peptidase Activity ◽  
Trichuris Muris ◽  
Hydrolysis Of

Excretory/secretory (E/S) material of Trichuris muris was found to contain 2 major peptidases, Mr 85 and 105 kDa, which degrade gelatin optimally at pH 6·0 in sodium dodecyl sulphate–polyacrylamide gels. The peptidases were inactivated diisopropylfluorophosphate, leupeptin and soybean trypsin inhibitor, but were unaffected by inhibitors of aspartic-, cysteine- and metallo-peptidases, indicating that they are serine peptidases. Both enzymes were detectable within 5 h after incubation of worms in culture medium and showed a time-dependent increase in levels. Neither peptidase was detected in worm extracts, suggesting that they are activated during or following secretion from worms. Live worms degraded radio-isotope labelled extracellular matrix protein substratum derived from mammalian cells. Aminopeptidase activities capable of catalysing hydrolysis of amino acyl aminomethylcoumarin (MCA) substrates and a Z-Phe-Arg-MCA-hydrolysing cysteine peptidase activity, were detected in extracts of adult worms but not in E/S material.

Kinetic characterization of hydrolysis of camel and bovine milk proteins by pancreatic enzymes

2008 ◽  
Vol 18 (12) ◽  
pp. 1097-1102 ◽  
Author(s):  
Maryam Salami ◽  
Reza Yousefi ◽  
Mohammad Reza Ehsani ◽  
Michèle Dalgalarrondo ◽  
Jean-Marc Chobert ◽  
...  
Keyword(s):  
Bovine Milk ◽  
Milk Proteins ◽  
Pancreatic Enzymes ◽  
Kinetic Characterization ◽  
Hydrolysis Of ◽  
Bovine Milk Proteins

scholarly journals Kinetic characterization of the acyl-enzyme mechanism for β-lactamase I

1988 ◽  
Vol 254 (3) ◽  
pp. 923-925 ◽  
Author(s):  
M T Martin ◽  
S G Waley
Keyword(s):  
Steady State ◽  
Rate Constants ◽  
Enzyme Mechanism ◽  
Beta Lactamase ◽  
Kinetic Characterization ◽  
Hydrolysis Of

beta-Lactamase I catalyses the hydrolysis of penicillins by an acyl-enzyme mechanism. A procedure was developed for determining the rate constants for the acylation and deacylation steps for the good substrates benzylpenicillin and phenoxymethylpenicillin; this depends on determining the fraction of enzyme that is present as acyl-enzyme in the steady state.

scholarly journals Characterization of the Prolyl Dipeptidyl Peptidase Gene (dppIV) from the Koji Mold Aspergillus oryzae

1998 ◽  
Vol 64 (12) ◽  
pp. 4809-4815 ◽  
Author(s):  
Agnès Doumas ◽  
Peter van den Broek ◽  
Michael Affolter ◽  
Michel Monod
Keyword(s):  
Aspergillus Oryzae ◽  
Polypeptide Chain ◽  
Wheat Gluten ◽  
Methylotrophic Yeast ◽  
Dipeptidyl Peptidase ◽  
Culture Broth ◽  
Peptidase Activity ◽  
Koji Mold ◽  
Hydrolysis Of

ABSTRACT The koji mold Aspergillus oryzae secretes a prolyl dipeptidyl peptidase (DPPIV) when the fungus is cultivated in a medium containing wheat gluten as the sole nitrogen and carbon source (MMWG). We cloned and sequenced the DPPIV gene from an A. oryzae library by using the A. fumigatus dppIVgene as a probe. Reverse transcriptase PCR experiments showed that theA. oryzae dppIV gene consists of two exons, the first of which is only 6 bp long. The gene encodes an 87.2-kDa polypeptide chain which is secreted into the medium as a 95-kDa glycoprotein. Introduction of this gene into A. oryzae leads to overexpression of prolyl dipeptidyl peptidase activity, while disruption of the gene abolishes all prolyl dipeptidyl peptidase activity in MMWG. The dppIV null mutants did not exhibit any change in phenotype other than the absence of prolyl dipeptidyl peptidase activity, suggesting that this activity is not essential. This loss of activity diminished the number of dipeptides and increased the number of larger peptides present in the MMWG culture broth. These effects were reversed by the addition of purified, recombinant DPPIV from the methylotrophic yeast expression vector Pichia pastoris. Our results suggest that the DPPIV enzyme may be of importance in industrial hydrolysis of what gluten-based substrates, which are rich in Pro residues.

scholarly journals Mutational effects on carbapenem hydrolysis of YEM-1, a new sub-class B2 metallo-β-lactamase from Yersinia mollaretii

2020 ◽  
Author(s):  
P. S. Mercuri ◽  
R. Esposito ◽  
S. Blétard ◽  
S. Di Costanzo ◽  
M. Perilli ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Catalytic Efficiency ◽  
The Other ◽  
Kinetic Characterization ◽  
Activity Profile ◽  
Gene Coding ◽  
Hydrolysis Of

ABSTRACTThe analysis of the genome sequence of Yersinia mollaretii (Y. mollaretii) ATCC 43969 indicates the presence of the blaYEM gene coding for YEM-1, a putative subclass B2 metallo-β-lactamase. The objectives of our work were to produce, purify and complete the kinetic characterization of YEM-1. Compared to the known subclass B2 metallo–β-lactamases, YEM-1 displayed a narrowest substrate profile since it is only able to hydrolyse imipenem with a high catalytic efficiency but not all the other carbapenems tested such as biapenem, meropenem, doripenem and ertapenem. A possible explanation of this peculiar activity profile is the presence of tyrosine 67 (loop L1), threonine 156 (loop L2) and serine 236 (loop L3) respectively. We showed that the substitution of Y67 broadened the activity profile of the enzyme for all carbapenems but still displayed a poor activity toward the other β-lactam classes.

Purification and characterization of an extracellular thiol-containing serine proteinase from Thermomyces lanuginosus

1992 ◽  
Vol 70 (2) ◽  
pp. 117-122 ◽  
Author(s):  
Sadiq Hasnain ◽  
Khosrow Adeli ◽  
Andrew C. Storer
Keyword(s):  
Molecular Mass ◽  
Serine Protease ◽  
Filamentous Fungus ◽  
Serine Proteinase ◽  
Thermomyces Lanuginosus ◽  
Kinetic Characterization ◽  
Purification And Characterization ◽  
Inhibitor Specificity ◽  
Protein Substrates

An extracellular protease produced by the filamentous fungus Thermomyces lanuginosus has been purified and characterized. The results indicate that the enzyme, which we have called humicolin, is a thiol-containing serine protease with a molecular mass of 38 000 kilodaltons. Secretion of humicolin, which is glycosylated, is tightly regulated by protein substrates. Kinetic characterization has revealed that humicolin activity is highly dependent upon the deprotonation of a group with a pKa of 6.6 and that the enzyme has a specificity for phenylalanine in the P1 position of the substrate.Key words: thiol-containing serine proteinase, characterization, kinetics, inhibitor, specificity.

scholarly journals Kinetic characterization of human butyrylcholinesterase mutants for the hydrolysis of cocaethylene

2014 ◽  
Vol 460 (3) ◽  
pp. 447-457 ◽  
Author(s):  
Shurong Hou ◽  
Max Zhan ◽  
Xirong Zheng ◽  
Chang-Guo Zhan ◽  
Fang Zheng
Keyword(s):  
Kinetic Modelling ◽  
Kinetic Characterization ◽  
In Vivo Tests ◽  
Hydrolysis Of ◽  
Human Butyrylcholinesterase

Catalytic parameters of butyrylcholinesterase and its mutants against cocaethylene have been characterized in comparison with those against cocaine, indicating that the mutants can efficiently metabolize cocaethylene, in addition to cocaine. Further in vivo tests and kinetic modelling support the indication.

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