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 Complete amino acid analysis of peptides and proteins after hydrolysis by a mixture of Sepharose-bound peptidases

1972 ◽  
Vol 129 (3) ◽  
pp. 695-701 ◽  
Author(s):  
H. P. J. Bennett ◽  
D. F. Elliott ◽  
B. E. Evans ◽  
P. J. Lowry ◽  
C. McMartin
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Standard Deviation ◽  
Quantitative Determination ◽  
Edman Degradation ◽  
Amino Acid Residues ◽  
Peptide Fragments ◽  
Enzyme Mixture ◽  
Acid Labile

Incubation with a mixture of Sepharose-bound peptidases was shown to result in the quantitative release of amino acids from certain peptides and S-aminoethylated proteins. Subtraction of the low background values of amino acids generated by the enzymes enables amino acid ratios of corticotrophin-(1–24)-tetracosapeptide to be determined with a standard deviation on repeat digestions of 3–5%. Good values were obtained for amino acids that are completely or partially destroyed on acid hydrolysis, i.e. tryptophan, tyrosine, serine, asparagine and glutamine. Experiments with peptides containing d-amino acids showed that the enzyme mixture is stereospecific and could therefore be used to detect the presence of d-residues in peptides. The enzyme mixture completely hydrolyses peptide fragments obtained after Edman degradation and should therefore be useful for determining sequences of peptides containing acid-labile amino acid residues. The activities of the bound enzymes were unaltered over a period of 7 months and they provide a simple, reproducible procedure for the quantitative determination of amino acids in peptides and proteins containing l-amino acids.

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 Comprehensive mutagenesis to identify amino acid residues contributing to the difference in thermostability between two originally thermostable ancestral proteins

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0258821
Author(s):  
Satoshi Akanuma ◽  
Minako Yamaguchi ◽  
Akihiko Yamagishi
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rational Design ◽  
Synergistic Effects ◽  
Amino Acid Sequences ◽  
Amino Acid Substitutions ◽  
Amino Acid Residues ◽  
Identify Amino Acid ◽  
The Difference ◽  
The Stability

Further improvement of the thermostability of inherently thermostable proteins is an attractive challenge because more thermostable proteins are industrially more useful and serve as better scaffolds for protein engineering. To establish guidelines that can be applied for the rational design of hyperthermostable proteins, we compared the amino acid sequences of two ancestral nucleoside diphosphate kinases, Arc1 and Bac1, reconstructed in our previous study. Although Bac1 is a thermostable protein whose unfolding temperature is around 100°C, Arc1 is much more thermostable with an unfolding temperature of 114°C. However, only 12 out of 139 amino acids are different between the two sequences. In this study, one or a combination of amino acid(s) in Bac1 was/were substituted by a residue(s) found in Arc1 at the same position(s). The best mutant, which contained three amino acid substitutions (S108D, G116A and L120P substitutions), showed an unfolding temperature more than 10°C higher than that of Bac1. Furthermore, a combination of the other nine amino acid substitutions also led to improved thermostability of Bac1, although the effects of individual substitutions were small. Therefore, not only the sum of the contributions of individual amino acids, but also the synergistic effects of multiple amino acids are deeply involved in the stability of a hyperthermostable protein. Such insights will be helpful for future rational design of hyperthermostable proteins.

Determination of the Complete Absolute Configuration of Petriellin A

2006 ◽  
Vol 59 (6) ◽  
pp. 407 ◽  
Author(s):  
Luigi Aurelio ◽  
Robert T. C. Brownlee ◽  
Jason Dang ◽  
Andrew B. Hughes ◽  
Gideon M. Polya
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Natural Product ◽  
Absolute Configuration ◽  
Structural Determination ◽  
Amino Acid Residues ◽  
The Common ◽  
The Absolute ◽  
Derivatives Of

We report the full structural determination of the depsipeptide petriellin A. The absolute configuration of the amino acid residues, N-methyl isoleucine and N-methyl threonine, have been determined by a combination of HPLC and TLC comparison of synthetic Marfey’s derivatives and Marfey’s derivatives of the natural product hydrolysate. The configuration of the chiral centres in these two N-methylated residues was found to be the same as those of the common unmethylated l-amino acids.

scholarly journals Amino Acids in Positions 48, 52, and 73 Differentiate the Substrate Specificities of the Highly Homologous Chlorocatechol 1,2-Dioxygenases CbnA and TcbC

2005 ◽  
Vol 187 (15) ◽  
pp. 5427-5436 ◽  
Author(s):  
Shenghao Liu ◽  
Naoto Ogawa ◽  
Toshiya Senda ◽  
Akira Hasebe ◽  
Kiyotaka Miyashita
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ralstonia Eutropha ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Sequence Comparisons ◽  
Substrate Specificities ◽  
High Conservation ◽  
The Difference

ABSTRACT Chlorocatechol 1,2-dioxygenase (CCD) is the first-step enzyme of the chlorocatechol ortho-cleavage pathway, which plays a central role in the degradation of various chloroaromatic compounds. Two CCDs, CbnA from the 3-chlorobenzoate-degrader Ralstonia eutropha NH9 and TcbC from the 1,2,4-trichlorobenzene-degrader Pseudomonas sp. strain P51, are highly homologous, having only 12 different amino acid residues out of identical lengths of 251 amino acids. But CbnA and TcbC are different in substrate specificities against dichlorocatechols, favoring 3,5-dichlorocatechol (3,5-DC) and 3,4-dichlorocatechol (3,4-DC), respectively. A study of chimeric mutants constructed from the two CCDs indicated that the N-terminal parts of the enzymes were responsible for the difference in the substrate specificities. Site-directed mutagenesis studies further identified the amino acid in position 48 (Leu in CbnA and Val in TcbC) as critical in differentiating the substrate specificities of the enzymes, which agreed well with molecular modeling of the two enzymes. Mutagenesis studies also demonstrated that Ile-73 of CbnA and Ala-52 of TcbC were important for their high levels of activity towards 3,5-DC and 3,4-DC, respectively. The importance of Ile-73 for 3,5-DC specificity determination was also shown with other CCDs such as TfdC from Burkholderia sp. NK8 and TfdC from Alcaligenes sp. CSV90 (identical to TfdC from R. eutropha JMP134), which convert 3,5-DC preferentially. Together with amino acid sequence comparisons indicating high conservation of Leu-48 and Ile-73 among CCDs, these results suggested that TcbC of strain P51 had diverged from other CCDs to be adapted to conversion of 3,4-DC.

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.

scholarly journals Determination of amino acids in human biological fluids by high-performance liquid chromatography: critical review

Amino Acids ◽  
2021 ◽  
Author(s):  
Grażyna Gałęzowska ◽  
Joanna Ratajczyk ◽  
Lidia Wolska
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
High Performance ◽  
Hplc Analysis ◽  
Limit Of Detection ◽  
Biological Fluids ◽  
Analytical Techniques ◽  
Epidemiological Studies ◽  
Preparation Conditions

AbstractThe quantitation and qualification of amino acids are most commonly used in clinical and epidemiological studies, and provide an excellent way of monitoring compounds in human fluids which have not been monitored previously, to prevent some diseases. Because of this, it is not surprising that scientific interest in evaluating these compounds has resurfaced in recent years and has precipitated the development of a multitude of new analytical techniques. This review considers recent developments in HPLC analytics on the basis of publications from the last few years. It helps to update and systematize knowledge in this area. Particular attention is paid to the progress of analytical methods, pointing out the advantages and drawbacks of the various techniques used for the preparation, separation and determination of amino acids. Depending on the type of sample, the preparation conditions for HPLC analysis change. For this reason, the review has focused on three types of samples, namely urine, blood and cerebrospinal fluid. Despite time-consuming sample preparation before HPLC analysis, an additional derivatization technique should be used, depending on the detection technique used. There are proposals for columns that are specially modified for amino acid separation without derivatization, but the limit of detection of the substance is less beneficial. In view of the fact that amino acid analyses have been performed for years and new solutions may generate increased costs, it may turn out that older proposals are much more advantageous.

scholarly journals Determination of Methylated Basic Amino Acids with the Amino Acid Analyzer

1973 ◽  
Vol 248 (7) ◽  
pp. 2387-2391 ◽  
Author(s):  
Gladys E. Deibler ◽  
Russell E. Martenson
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Basic Amino Acids ◽  
Amino Acid Analyzer

scholarly journals Determination of free amino acids in plants by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)

2015 ◽  
Vol 7 (18) ◽  
pp. 7574-7581 ◽  
Author(s):  
Magdalena M. Dziągwa-Becker ◽  
Jose M. Marin Ramos ◽  
Jakub K. Topolski ◽  
Wiesław A. Oleszek
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Liquid Chromatography ◽  
Amino Acid ◽  
Tandem Mass Spectrometry ◽  
Free Amino ◽  
Tandem Mass ◽  
Amino Acid Determination ◽  
Acid Determination

Free amino acid determination in plants by LC-MS/MS.

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