scholarly journals Subsite specificity (S3, S2, S1', S2' and S3') of oligopeptidase B from Trypanosoma cruzi and Trypanosoma brucei using fluorescent quenched peptides: comparative study and identification of specific carboxypeptidase activity

2003 ◽  
Vol 373 (3) ◽  
pp. 933-939 ◽  
Author(s):  
Jefferson P. HEMERLY ◽  
Vitor OLIVEIRA ◽  
Elaine DEL NERY ◽  
Rory E. MORTY ◽  
Norma W. ANDREWS ◽  
...  
Keyword(s):  
Amino Acids ◽  
Trypanosoma Cruzi ◽  
Trypanosoma Brucei ◽  
Peptide Bond ◽  
Minimum Size ◽  
Amino Acid Residues ◽  
Basic Amino Acids ◽  
Oligopeptidase B ◽  
Work Supports ◽  
Micromolar Range

We characterized the extended substrate binding site of recombinant oligopeptidase B enzymes from Trypanosoma cruzi (Tc-OP) and Trypanosoma brucei (Tb-OP), evaluating the specificity of their S3, S2, S1′, S2′ and S3′ subsites. Five series of internally quenched fluorescent peptides based on the substrate Abz-AGGRGAQ-EDDnp [where Abz is o-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine] were designed to contain amino acid residues with side chains of a minimum size, and each residue position of this substrate was modified. Synthetic peptides of different lengths derived from the human kininogen sequence were also examined, and peptides of up to 17 amino acids were found to be hydrolysed by Tc-OP and Tb-OP. These two oligopeptidases were essentially arginyl hydrolases, since for all peptides examined the only cleavage site was the Arg–Xaa bond. We also demonstrated that Tc-OP and Tb-OP have a very specific carboxypeptidase activity for basic amino acids, which depends on the presence of at least of a pair of basic amino acids at the C-terminal end of the substrate. The peptide with triple Arg residues (Abz-AGRRRAQ-EDDnp) was an efficient substrate for Tc-OP and Tb-OP: the Arg–Ala peptide bond was cleaved first and then two C-terminal Arg residues were successively removed. The S1′ subsite seems to be an important determinant of the specificity of both enzymes, showing a preference for Tyr, Ser, Thr and Gln as hydrogen donors. The presence of these amino acids at P1′ resulted in substrates that were hydrolysed with Km values in the sub-micromolar range. Taken together, this work supports the view that oligopeptidase B is a specialized protein-processing enzyme with a specific carboxypeptidase activity. Excellent substrates were obtained for Tb-OP and Tc-OP (Abz-AMRRTISQ-EDDnp and Abz-AHKRYSHQ-EDDnp respectively), which were hydrolysed with remarkably high kcat and low Km values.

scholarly journals The specificity of bovine spleen cathepsin S. A comparison with rat liver cathepsins L and B

1989 ◽  
Vol 264 (2) ◽  
pp. 475-481 ◽  
Author(s):  
D Brömme ◽  
A Steinert ◽  
S Friebe ◽  
S Fittkau ◽  
B Wiederanders ◽  
...  
Keyword(s):  
Rat Liver ◽  
Cathepsin L ◽  
Peptide Bond ◽  
Cathepsin S ◽  
Cleavage Sites ◽  
Amino Acid Residues ◽  
Peptide Substrates ◽  
Hydrophobic Residues ◽  
Micromolar Range ◽  
Better Than

The peptide-bond-specificity of bovine spleen cathepsin S in the cleavage of the oxidized insulin B-chain and peptide methylcoumarylamide substrates was investigated and the results are compared with those obtained with rat liver cathepsins L and B. Major cleavage sites in the oxidized insulin B-chain generated by cathepsin S are the bonds Glu13-Ala14, Leu17-Val18 and Phe23-Tyr26; minor cleavage sites are the bonds Asn3-Gln4, Ser9-His10 and Leu15-Tyr16. The bond-specificity of this proteinase is in part similar to the specificities of cathepsin L and cathepsin N. Larger differences are discernible in the reaction with synthetic peptide substrates. Cathepsin S prefers smaller neutral amino acid residues in the subsites S2 and S3, whereas cathepsin L efficiently hydrolyses substrates with bulky hydrophobic residues in the P2 and P3 positions. The results obtained from inhibitor studies differ somewhat from those based on substrates. Z-Phe-Ala-CH2F (where Z- represents benzyloxycarbonyl-) is a very potent time-dependent inhibitor for cathepsin S, and inhibits this proteinase 30 times more efficiently than it does cathepsin L and about 300 times better than it does cathepsin B. By contrast, the peptidylmethanes Z-Val-Phe-CH3 and Z-Phe-Lys(Z)-CH3 inhibit competitively both cathepsin S and cathepsin L in the micromolar range.

scholarly journals The Uptake and Metabolism of Amino Acids, and Their Unique Role in the Biology of Pathogenic Trypanosomatids

Pathogens ◽  
2018 ◽  
Vol 7 (2) ◽  
pp. 36 ◽  
Author(s):  
Letícia Marchese ◽  
Janaina Nascimento ◽  
Flávia Damasceno ◽  
Frédéric Bringaud ◽  
Paul Michels ◽  
...  
Keyword(s):  
Amino Acids ◽  
Trypanosoma Cruzi ◽  
Trypanosoma Brucei ◽  
Life Cycles ◽  
Unique Role ◽  
Physical Chemical ◽  
Intermediate Metabolites ◽  
Natural Hosts ◽  
Uptake And Metabolism ◽  
Genus Leishmania

Trypanosoma brucei, as well as Trypanosoma cruzi and more than 20 species of the genus Leishmania, form a group of flagellated protists that threaten human health. These organisms are transmitted by insects that, together with mammals, are their natural hosts. This implies that during their life cycles each of them faces environments with different physical, chemical, biochemical, and biological characteristics. In this work we review how amino acids are obtained from such environments, how they are metabolized, and how they and some of their intermediate metabolites are used as a survival toolbox to cope with the different conditions in which these parasites should establish the infections in the insects and mammalian hosts.

Peptide synthesis catalyzed by native proteinase K in water-miscible organic solvents with low water content

1989 ◽  
Vol 54 (7) ◽  
pp. 2027-2041 ◽  
Author(s):  
Václav Čeřovský ◽  
Karel Martinek
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Water Content ◽  
Organic Solvents ◽  
Peptide Bond ◽  
Component Composition ◽  
Proteinase K ◽  
Amino Acid Residues ◽  
Negative Effect ◽  
Hydrophobic Amino Acids

Reaction of Ac-Tyr-OEt with HBr.Gly-NH2, catalyzed by free proteinase K in various water-miscible organic solvents in the presence of triethylamine and 5 vol.% of water, was studied. Some aliphatic alcohols and acetonitrile proved to be suitable solvents. The effect of water content (2%-20%) on the synthesis of Ac-Tyr-Gly-NH2 was studied using acetonitrile as solvent. Lowering of the water content to 5% or 2% led to almost 100% yield of the desired dipeptide; higher water content accelerated the reaction, reducing at the same time the yield of Ac-Tyr-Gly-NH2 due to the concurrent hydrolysis of the ester Ac-Tyr-OEt. No reaction was observed in the absence of base (triethylamine), whereas an excess of base only retarded the reaction. The enzyme is capable of catalyzing the peptide bond synthesis with N-acylamino acids or N-acyl peptides as acylating components, which may contain all types of L-amino acid residues (except Pro) in the P1 position. However, the peptide bond synthesis depends strongly on the amino component composition, particularly on the amino acid residue in the P'1 position. Only amides of glycine and of hydrophilic amino acids were acylated with Ac-Tyr-OEt; amides of hydrophobic amino acids enter the reaction only reluctantly or not at all. The presence of Leu or Phe in position P'2 and Leu in position P'3 has not so negative effect on acylation of the amino component as has its presence in the P'1 position. The choice of protecting groups for the α-carboxyl of the amino component is restricted only to amide and in some cases its undesired enzymatic removal was observed. Unprotected peptides seem to be suitable amino components.

scholarly journals Differential Inhibition of Human and Trypanosome Ubiquitin E1S by TAK-243 Offers Possibilities for Parasite Selective Inhibitors

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
D. Roeland Boer ◽  
Marie-José Bijlmakers
Keyword(s):  
Amino Acids ◽  
Trypanosoma Cruzi ◽  
Trypanosoma Brucei ◽  
Human African Trypanosomiasis ◽  
Potent Inhibitor ◽  
Model System ◽  
Differential Inhibition ◽  
Protein Ubiquitination ◽  
Related Compounds

Abstract Novel strategies to target Trypanosoma brucei, Trypanosoma cruzi and Leishmania are urgently needed to generate better and safer drugs against Human African Trypanosomiasis, Chagas disease and Leishmaniasis, respectively. Here, we investigated the feasibility of selectively targeting in trypanosomatids the ubiquitin E1 activating enzyme (UBA1), an essential eukaryotic protein required for protein ubiquitination. Trypanosomatids contain two UBA1 genes in contrast to mammals and yeast that only have one, and using T. brucei as a model system, we show that both are active in vitro. Surprisingly, neither protein is inhibited by TAK-243, a potent inhibitor of human UBA1. This resistance stems from differences with the human protein at key amino acids, which includes a residue termed the gatekeeper because its mutation in E1s leads to resistance to TAK-243 and related compounds. Importantly, our results predict that trypanosomatid selective UBA1 inhibition is feasible and suggest ways to design novel compounds to achieve this.

scholarly journals Artemisinins Inhibit Trypanosoma cruzi and Trypanosoma brucei rhodesiense In Vitro Growth

2007 ◽  
Vol 51 (5) ◽  
pp. 1852-1854 ◽  
Author(s):  
Yuliya V. Mishina ◽  
Sanjeev Krishna ◽  
Richard K. Haynes ◽  
John C. Meade
Keyword(s):  
Trypanosoma Cruzi ◽  
Atpase Activity ◽  
Trypanosoma Brucei ◽  
Mode Of Action ◽  
In Vitro Growth ◽  
Trypanosoma Brucei Rhodesiense ◽  
Calcium Dependent ◽  
Membrane Pumps ◽  
Micromolar Range

ABSTRACT Artemisinin compounds inhibit in vitro growth of cultured Trypanosoma cruzi and Trypanosoma brucei rhodesiense at concentrations in the low micromolar range. Artemisinin also inhibits calcium-dependent ATPase activity in T. cruzi membranes, suggesting a mode of action via membrane pumps. Artemisinins merit further investigation as chemotherapeutic options for these pathogens.

Does the polyamine carrier system absorb basic amino acids?

2001 ◽  
Vol 120 (5) ◽  
pp. A142-A142
Author(s):  
J GASKEY ◽  
E SEIDEL
Keyword(s):  
Amino Acids ◽  
Carrier System ◽  
Basic Amino Acids

The Kinetics of Inhibition of the Action of Thrombin by the Fibrinopeptides Formed during the Clotting of Fibrinogen

1966 ◽  
Vol 16 (01/02) ◽  
pp. 277-295 ◽  
Author(s):  
A Silver ◽  
M Murray
Keyword(s):  
Amino Acids ◽  
Competitive Inhibition ◽  
Amorphous Material ◽  
Peptide Bond ◽  
Initial Reaction ◽  
Reaction Products ◽  
Coagulation Mechanism ◽  
Kinetics Of ◽  
Kinetics Of Inhibition ◽  
Burk Plot

SummaryVarious investigators have separated the coagulation products formed when fibrinogen is clotted with thrombin and identified fibrinopeptides A and B. Two other peaks are observed in the chromatogram of the products of coagulation, but these have mostly been dismissed by other workers. They have been identified by us as amino acids, smaller peptides and amorphous material (37). We have re-chromatographed these peaks and identified several amino acids. In a closed system of fibrinogen and thrombin, the only reaction products should be fibrin and peptide A and peptide B. This reasoning has come about because thrombin has been reported to be specific for the glycyl-arginyl peptide bond. It is suggested that thrombin also breaks other peptide linkages and the Peptide A and Peptide B are attacked by thrombin to yield proteolytic products. Thrombin is therefore probably not specific for the glycyl-arginyl bond but will react on other linkages as well.If the aforementioned is correct then the fibrinopeptides A and B would cause an inhibition with the coagulation mechanism itself. We have shown that an inhibition does occur. We suggest that there is an autoinhibition to the clotting mechanism that might be a control mechanism in the human body.The experiment was designed for coagulation to occur under controlled conditions of temperature and time. Purified reactants were used. We assembled an apparatus to record visually the speed of the initial reaction, the rate of the reaction, and the density of the final clot formed after a specific time.The figures we derived made available to us data whereby we could calculate and plot the information to show the mechanism and suggest that such an inhibition does exist and also further suggest that it might be competitive.In order to prove true competitive inhibition it is necessary to fulfill the criteria of the Lineweaver-Burk plot. This has been done. We have also satisfied other criteria of Dixon (29) and Bergman (31) that suggest true competitive inhibition.

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