scholarly journals The Effect of a Peptide Substrate Containing an Unnatural Branched Amino Acid on Chymotrypsin Activity

Processes ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 242
Author(s):  
Yuuki Yamawaki ◽  
Tomoki Yufu ◽  
Tamaki Kato
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Unnatural Amino Acids ◽  
Side Chain ◽  
Low Molecular Weight ◽  
Amino Acid Residues ◽  
Phase Synthesis ◽  
Reaction Velocity ◽  
Natural Amino Acids

7-Amino-4-methylcoumarin (AMC) is a low molecular weight fluorescent probe that can be attached to a peptide to enable the detection of specific proteases, such as chymotrypsin, expressed in certain diseases. Because this detection depends on the specificity of the protease toward the peptidyl AMC, the development of specific substrates is required. To investigate the specificity of chymotrypsin, peptidyl AMC compounds incorporating four different amino acid residues were prepared by liquid-phase synthesis. Two unnatural amino acids, 2-amino-4-ethylhexanoic acid (AEH) and cyclohexylalanine (Cha), were used to investigate the substrate specificity as these amino acids have structures different from natural amino acids. AEH was synthesized using diethyl acetamidemalonate as a starting material. The substrate containing Cha had high hydrophobicity and showed a high reaction velocity with chymotrypsin. Although the AEH substrate with a branched side chain had high hydrophobicity, it showed a low reaction velocity. The substrate containing the aromatic amino acid phenylalanine was less hydrophobic than the Cha and AEH substrates, but chymotrypsin showed the highest specificity for this compound. These results demonstrated that the substrate specificity of chymotrypsin is not only affected by the hydrophobicity and aromaticity, but also by the structural expanse of amino acid residues in the substrate.

scholarly journals A Novelmeso-Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum: Overexpression, Characterization, and Potential for d-Amino Acid Synthesis

2012 ◽  
Vol 78 (24) ◽  
pp. 8595-8600 ◽  
Author(s):  
Xiuzhen Gao ◽  
Xi Chen ◽  
Weidong Liu ◽  
Jinhui Feng ◽  
Qiaqing Wu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Enantiomeric Excess ◽  
Acid Synthesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Gene Encoding ◽  
Amino Acid Synthesis ◽  
Content Type

ABSTRACTmeso-Diaminopimelate dehydrogenase (meso-DAPDH) is an NADP+-dependent enzyme which catalyzes the reversible oxidative deamination on thed-configuration ofmeso-2,6-diaminopimelate to producel-2-amino-6-oxopimelate. In this study, the gene encoding ameso-diaminopimelate dehydrogenase fromSymbiobacterium thermophilumwas cloned and expressed inEscherichia coli. In addition to the native substratemeso-2,6-diaminopimelate, the purified enzyme also showed activity towardd-alanine,d-valine, andd-lysine. This enzyme catalyzed the reductive amination of 2-keto acids such as pyruvic acid to generated-amino acids in up to 99% conversion and 99% enantiomeric excess. Sincemeso-diaminopimelate dehydrogenases are known to be specific tomeso-2,6-diaminopimelate, this is a unique wild-typemeso-diaminopimelate dehydrogenase with a more relaxed substrate specificity and potential ford-amino acid synthesis. The enzyme is the most stablemeso-diaminopimelate dehydrogenase reported to now. Two amino acid residues (F146 and M152) in the substrate binding sites ofS. thermophilum meso-DAPDH different from the sequences of other knownmeso-DAPDHs were replaced with the conserved amino acids in othermeso-DAPDHs, and assay of wild-type and mutant enzyme activities revealed that F146 and M152 are not critical in determining the enzyme's substrate specificity. The high thermostability and relaxed substrate profile ofS. thermophilum meso-DAPDH warrant it as an excellent starting enzyme for creating effectived-amino acid dehydrogenases by protein engineering.

Solid-phase synthesis and utilization of side-chain reactive unnatural amino acids

2003 ◽  
Vol 44 (46) ◽  
pp. 8403-8406 ◽  
Author(s):  
Martin J. O'Donnell ◽  
Jordi Alsina ◽  
William L. Scott
Keyword(s):  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
Side Chain ◽  
Phase Synthesis

Gram-Scale Solution-Phase Synthesis of Heptapeptide Side Chain of Teixobactin

Synlett ◽  
2019 ◽  
Vol 30 (20) ◽  
pp. 2268-2272 ◽  
Author(s):  
Sangeetha Donikela ◽  
Kiranmai Nayani ◽  
Vishnuvardhan Nomula ◽  
Prathama S. Mainkar ◽  
Srivari Chandrasekhar
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Natural Product ◽  
Solution Phase ◽  
Side Chain ◽  
Phase Synthesis ◽  
Solution Phase Synthesis ◽  
Efficient Coupling ◽  
Scalable Synthesis

We report herein a scalable synthesis of linear heptapeptide side chain of the depsipeptide natural product teixobactin through solution phase. The synthesis of heptapeptide was achieved through an efficient coupling of suitably protected tripeptide and tetrapeptide comprising of three d-amino acids and four usual l-amino acid subunits.

scholarly journals Roles of Residues Cys69, Asn104, Phe160, Gly232, Ser237, and Asp240 in Extended-Spectrum β-Lactamase Toho-1

2010 ◽  
Vol 55 (1) ◽  
pp. 284-290 ◽  
Author(s):  
Akiko Shimizu-Ibuka ◽  
Mika Oishi ◽  
Shoko Yamada ◽  
Yoshikazu Ishii ◽  
Kiyoshi Mura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Binding Site ◽  
Kinetic Properties ◽  
Amino Acid Residues ◽  
Class A ◽  
Extended Spectrum ◽  
Hydrolysis Of

ABSTRACTToho-1, which is also designated CTX-M-44, is an extended-spectrum class A β-lactamase that has high activity toward cefotaxime. In this study, we investigated the roles of residues suggested to be critical for the substrate specificity expansion of Toho-1 in previous structural analyses. Six amino acid residues were replaced one by one with amino acids that are often observed in the corresponding position of non-extended-spectrum β-lactamases. The mutants produced inEscherichia colistrains were analyzed both for their kinetic properties and their effect on drug susceptibilities. The results indicate that the substitutions of Asn104 and Ser237 have certain effects on expansion of substrate specificity, while those of Cys69 and Phe160 have less effect, and that of Asp240 has no effect on the hydrolysis of any substrates tested. Gly232, which had been assumed to increase the flexibility of the substrate binding site, was revealed not to be critical for the expansion of substrate specificity of this enzyme, although this substitution resulted in deleterious effects on expression and stability of the enzyme.

Recent advances and concepts in substrate specificity determination of proteases using tailored libraries of fluorogenic substrates with unnatural amino acids

2015 ◽  
Vol 396 (4) ◽  
pp. 329-337 ◽  
Author(s):  
Wioletta Rut ◽  
Paulina Kasperkiewicz ◽  
Anna Byzia ◽  
Marcin Poreba ◽  
Katarzyna Groborz ◽  
...  
Keyword(s):  
Amino Acids ◽  
Substrate Specificity ◽  
Phage Display ◽  
Unnatural Amino Acids ◽  
Fluorogenic Substrates ◽  
Recent Advances ◽  
Positional Scanning ◽  
Binding Pockets ◽  
Natural Amino Acids

Abstract Substrate specificity of proteases can be determined using several methods among which the most frequently used are positional scanning library, proteomics and phage display. Classic approaches can deliver information about preferences for natural amino acids in binding pockets of virtually all proteases. However, recent studies demonstrate the ability to obtain much more information by application of unnatural amino acids to positional scanning library approaches. This knowledge can be used for the design of more active and specific substrates, inhibitors and activity based probes. In this minireview we describe recent strategies and concepts for the design and application of fluorogenic substrates library tailored for exopeptidases and endopeptidases.

Synthesis and evaluation of chiral β-amino acid-based low-molecular-weight organogelators possessing a methyl/trifluoromethyl side chain

2019 ◽  
Vol 43 (7) ◽  
pp. 2882-2887 ◽  
Author(s):  
Koichi Kodama ◽  
Ryuta Kawamata ◽  
Takuji Hirose
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Supramolecular Structures ◽  
Side Chain ◽  
Low Molecular Weight ◽  
Side Chains ◽  
Low Molecular Weight Organogelators

Impacts of side-chains and chirality of organogelators derived from β-amino acids are described with their supramolecular structures.

scholarly journals Kinetics and Substrate Specificity of Membrane-Reconstituted Peptide Transporter DtpT ofLactococcus lactis

2000 ◽  
Vol 182 (9) ◽  
pp. 2530-2535 ◽  
Author(s):  
Gang Fang ◽  
Wil N. Konings ◽  
Bert Poolman
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Free Amino ◽  
Peptide Transport ◽  
Peptide Transporter ◽  
Amino Acid Residues ◽  
Peptide Bonds ◽  
Carboxyl Terminal ◽  
Affinity Interaction

ABSTRACT The peptide transport protein DtpT of Lactococcus lactis was purified and reconstituted into detergent-destabilized liposomes. The kinetics and substrate specificity of the transporter in the proteoliposomal system were determined, using Pro-[14C]Ala as a reporter peptide in the presence of various peptides or peptide mimetics. The DtpT protein appears to be specific for di- and tripeptides, with the highest affinities for peptides with at least one hydrophobic residue. The effect of the hydrophobicity, size, or charge of the amino acid was different for the amino- and carboxyl-terminal positions of dipeptides. Free amino acids, ω-amino fatty acid compounds, or peptides with more than three amino acid residues do not interact with DtpT. For high-affinity interaction with DtpT, the peptides need to have free amino and carboxyl termini, amino acids in the l configuration, andtrans-peptide bonds. Comparison of the specificity of DtpT with that of the eukaryotic homologues PepT1 and PepT2 shows that the bacterial transporter is more restrictive in its substrate recognition.

Schistosomal Sulfotransferase Interaction with Oxamniquine Involves Hybrid Mechanism of Induced-fit and Conformational Selection

2020 ◽  
Vol 16 (4) ◽  
pp. 451-459 ◽  
Author(s):  
Fortunatus C. Ezebuo ◽  
Ikemefuna C. Uzochukwu
Keyword(s):  
Molecular Dynamics ◽  
Amino Acid ◽  
Binding Energy ◽  
Binding Site ◽  
Conformational Dynamics ◽  
Side Chain ◽  
Amino Acid Residues ◽  
Conformational Selection ◽  
Hybrid Mechanism ◽  
Dynamics Simulations

Background: Sulfotransferase family comprises key enzymes involved in drug metabolism. Oxamniquine is a pro-drug converted into its active form by schistosomal sulfotransferase. The conformational dynamics of side-chain amino acid residues at the binding site of schistosomal sulfotransferase towards activation of oxamniquine has not received attention. Objective: The study investigated the conformational dynamics of binding site residues in free and oxamniquine bound schistosomal sulfotransferase systems and their contribution to the mechanism of oxamniquine activation by schistosomal sulfotransferase using molecular dynamics simulations and binding energy calculations. Methods: Schistosomal sulfotransferase was obtained from Protein Data Bank and both the free and oxamniquine bound forms were subjected to molecular dynamics simulations using GROMACS-4.5.5 after modeling it’s missing amino acid residues with SWISS-MODEL. Amino acid residues at its binding site for oxamniquine was determined and used for Principal Component Analysis and calculations of side-chain dihedrals. In addition, binding energy of the oxamniquine bound system was calculated using g_MMPBSA. Results: The results showed that binding site amino acid residues in free and oxamniquine bound sulfotransferase sampled different conformational space involving several rotameric states. Importantly, Phe45, Ile145 and Leu241 generated newly induced conformations, whereas Phe41 exhibited shift in equilibrium of its conformational distribution. In addition, the result showed binding energy of -130.091 ± 8.800 KJ/mol and Phe45 contributed -9.8576 KJ/mol. Conclusion: The results showed that schistosomal sulfotransferase binds oxamniquine by relying on hybrid mechanism of induced fit and conformational selection models. The findings offer new insight into sulfotransferase engineering and design of new drugs that target sulfotransferase.

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