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 Determination of three amino acids causing alteration of proteolytic activities of staphylococcal glutamyl endopeptidases

2009 ◽  
Vol 390 (3) ◽  
Author(s):  
Takayuki K. Nemoto ◽  
Toshio Ono ◽  
Yu Shimoyama ◽  
Shigenobu Kimura ◽  
Yuko Ohara-Nemoto
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protease Activity ◽  
Binding Pocket ◽  
Catalytic Triad ◽  
Amino Acid Residues ◽  
Proteolytic Activities ◽  
Hydrophobic Amino Acids ◽  
The Difference

Abstract Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus warneri secrete glutamyl endopeptidases, designated GluV8, GluSE, and GluSW, respectively. The order of their protease activities is GluSE<GluSW<<GluV8. In the present study, we investigated the mechanism that causes these differences. Expression of chimeric proteins between GluV8 and GluSE revealed that the difference is primarily attributed to amino acid residues 170–195, which define the intrinsic protease activity, and additionally to residues 119–169, which affect the proteolytic sensitivity. Among nine substitutions present in residues 170–195 of the three proteases, the substitutions at positions 185, 188, and 189 were responsible for the changes in their activities, and the combination of W185, V188, and P189, which naturally occurs in GluV8, exerts the highest protease activity. W185 and P189 were indispensable for full activity, but V188 could be replaced by hydrophobic amino acids. These three amino acid residues appear to create a substrate-binding pocket together with the catalytic triad and the N-terminal V1, and therefore define the K m values of the proteases. We also describe a method to produce a chimeric form of GluSE and GluV8 that is resistant to proteolysis, and therefore possesses 4-fold higher activity than the wild-type recombinant GluV8.

scholarly journals Identification of Bacillus thuringiensisDelta-Endotoxin Cry1C Domain III Amino Acid Residues Involved in Insect Specificity

1999 ◽  
Vol 65 (10) ◽  
pp. 4369-4374 ◽  
Author(s):  
Ruud A. de Maagd ◽  
Petra Bakker ◽  
Nikola Staykov ◽  
Stefan Dukiandjiev ◽  
Willem Stiekema ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Spodoptera Exigua ◽  
Specific Activity ◽  
Beet Armyworm ◽  
Amino Acid Residues ◽  
Optimal Activity ◽  
Amino Acid Insertion ◽  
Negative Effect ◽  
Domain Iii

ABSTRACT Cry1C domain III amino acid residues involved in specificity for beet armyworm (Spodoptera exigua) were identified. For this purpose, intradomain III hybrids between Cry1E (nontoxic) and Cry1E-Cry1C hybrid G27 (toxic) were made. Crossover points of these hybrids defined six sequence blocks containing between 1 and 19 of the amino acid differences between Cry1E and G27. Blocks B, C, D, and E of G27 were shown to be required for optimal activity against S. exigua. Block E was also required for optimal activity against the tobacco hornworm (Manduca sexta), whereas block D had a negative effect on toxicity for this insect. The mutagenesis of individual amino acids in block B identified Trp-476 as the only amino acid in this block essential, although not sufficient by itself, for full S. exigua activity. In block D, we identified a seven-amino-acid insertion in G27 that was not in Cry1E. The deletion of either one of two groups of four consecutive amino acids in this insertion completely abolished activity against S. exiguabut resulted in higher activity against M. sexta. Alanine substitutions of the first group had little effect on toxicity, whereas alanine substitutions of the second group had the same effect as its deletion. These results identify groups of amino acids as well as some individual residues in Cry1C domain III, which are strongly involved inS. exigua-specific activity as well as sometimes involved in M. sexta-specific activity.

13C nuclear magnetic resonance study of cyclodipeptides containing 13C enriched hydrophobic amino acids

1980 ◽  
Vol 45 (2) ◽  
pp. 482-490 ◽  
Author(s):  
Jaroslav Vičar ◽  
François Piriou ◽  
Pierre Fromageot ◽  
Karel Bláha ◽  
Serge Fermandjian
Keyword(s):  
Amino Acids ◽  
Nuclear Magnetic Resonance ◽  
Nuclear Magnetic Resonance Study ◽  
Coupling Constants ◽  
Side Chain ◽  
Amino Acid Residues ◽  
Magnetic Resonance Study ◽  
Side Chain Conformation ◽  
13C Nuclear Magnetic Resonance ◽  
Hydrophobic Amino Acids

The diastereoisomeric pairs of cyclodipeptides cis- and trans-cyclo(Ala-Ala), cyclo(Ala-Phe), cyclo(Val-Val) and cyclo(Leu-Leu) containing 85% 13C enriched amino-acid residues were synthesized and their 13C-13C coupling constants were measured. The combination of 13C-13C and 1H-1H coupling constants enabled to estimate unequivocally the side chain conformation of the valine and leucine residues.

The C-terminal sequence of amino acid residues of κ-casein isolated from buffalo's milk

1967 ◽  
Vol 34 (1) ◽  
pp. 85-88 ◽  
Author(s):  
M. H. Abd El-Salam ◽  
W. Manson
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Peptide Chain ◽  
Amino Acid Residues ◽  
Terminal Sequence ◽  
Carboxypeptidase A ◽  
Phosphorous Content ◽  
Single Peptide

SummaryWhen κ-casein from buffalo's milk was treated with carboxypeptidase A (EC 3. 4. 2. 1),4 amino acids, valine, threonine, serine and alanine were released from the protein in a manner consistent with the view that they originate in the C-terminal sequence of a single peptide chain. The amounts produced suggest a minimum molecular weight for buffalo κ-casein of approximately 17000, in agreement with the value calculated from the phosphorous content on the basis of the presence of 2 phosphorus atoms/molecule. A comparison is made with the C-terminal sequence reported for bovine κ-casein.

Structural characterization of folded and extended conformations in peptides containing γ amino acids with proteinogenic side chains: crystal structures of γn, (αγ)n and γγδγ sequences

2015 ◽  
Vol 39 (5) ◽  
pp. 3319-3326 ◽  
Author(s):  
Madhusudana M. B. Reddy ◽  
K. Basuroy ◽  
S. Chandrappa ◽  
B. Dinesh ◽  
B. Vasantha ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Crystal Structures ◽  
Structural Characterization ◽  
Side Chains ◽  
Amino Acid Residues

γn amino acid residues can be incorporated into structures in γn and hybrid sequences containing folded and extended α and δ residues.

Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus

1989 ◽  
Vol 9 (1) ◽  
pp. 83-91
Author(s):  
S Miyazawa ◽  
T Osumi ◽  
T Hashimoto ◽  
K Ohno ◽  
S Miura ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Coenzyme A ◽  
Terminal Region ◽  
Proteinase K ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Targeting Signal ◽  
Acyl Coenzyme A

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

Consistency and Change

2019 ◽  
Author(s):  
Alan Kelly
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Living Organism ◽  
Peptide Bond ◽  
The Body ◽  
Blood Proteins ◽  
Genes Encoding ◽  
Fundamental Phenomenon ◽  
P Proteins

Proteins are, in my view, the most impressive molecules in food. They influence the texture, crunch, chew, flow, color, flavor, and nutritional quality of food. Not only that, but they can radically change their properties and how they behave depending on the environment and, critically for food, in response to processes like heating. Even when broken down into smaller components they are important, for example giving cheese many of its critical flavor notes. Indeed, I would argue that perhaps the most fundamental phenomenon we encounter in cooking or processing food is the denaturation of proteins, as will be explained shortly. Beyond food, the value of proteins and their properties is widespread across biology. Many of the most significant molecules in our body and that of any living organism (including plants and animals) are proteins. These include those that make hair and skin what they are, as well as the hemoglobin that transports oxygen around the body in our blood. Proteins are built from amino acids, a family of 20 closely related small molecules, which all have in chemical terms the same two ends (chemically speaking, an amino end and an acidic end, hence the name) but differ in the middle. This bit in the middle varies from amino acid to amino acid, from simple (a hydrogen atom in the case of glycine, the simplest amino acid) to much more complex structures. Amino acids can link up very neatly, as the amino end of one can form a bond (called a peptide bond) with the acid end of another, and so forth, so that chains of amino acids are formed that, when big enough (more than a few dozen amino acids), we call proteins. Our bodies produce thousands of proteins for different functions, and the instructions for which amino acids combine to make which proteins are essentially what the genetic code encrypted in our DNA specifies. We hear a lot about our genes encoding the secrets of life, but what that code spells is basically P-R-O-T-E-I-N. Yes, these are very important molecules!

scholarly journals SLC6A14, a Pivotal Actor on Cancer Stage: When Function Meets Structure

2019 ◽  
Vol 24 (9) ◽  
pp. 928-938 ◽  
Author(s):  
Luca Palazzolo ◽  
Chiara Paravicini ◽  
Tommaso Laurenzi ◽  
Sara Adobati ◽  
Simona Saporiti ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Amino Acids ◽  
Amino Acid ◽  
Molecular Dynamics Simulations ◽  
Electrostatic Interactions ◽  
Amino Acid Residues ◽  
Dibasic Amino Acid ◽  
Novel Target ◽  
Function Relationship ◽  
Dynamics Simulations

SLC6A14 (ATB0,+) is a sodium- and chloride-dependent neutral and dibasic amino acid transporter that regulates the distribution of amino acids across cell membranes. The transporter is overexpressed in many human cancers characterized by an increased demand for amino acids; as such, it was recently acknowledged as a novel target for cancer therapy. The knowledge on the molecular mechanism of SLC6A14 transport is still limited, but some elegant studies on related transporters report the involvement of the 12 transmembrane α-helices in the transport mechanism, and describe structural rearrangements mediated by electrostatic interactions with some pivotal gating residues. In the present work, we constructed a SLC6A14 model in outward-facing conformation via homology modeling and used molecular dynamics simulations to predict amino acid residues critical for substrate recognition and translocation. We docked the proteinogenic amino acids and other known substrates in the SLC6A14 binding site to study both gating regions and the exposed residues involved in transport. Interestingly, some of these residues correspond to those previously identified in other LeuT-fold transporters; however, we could also identify a novel relevant residue with such function. For the first time, by combined approaches of molecular docking and molecular dynamics simulations, we highlight the potential role of these residues in neutral amino acid transport. This novel information unravels new aspects of the human SLC6A14 structure–function relationship and may have important outcomes for cancer treatment through the design of novel inhibitors of SLC6A14-mediated transport.

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