Effect of peptide bond cleavage on the racemization of amino acid residues in proteins

1987 ◽  
Vol 35 (5) ◽  
pp. 661-667 ◽  
Author(s):  
Remy Liardon ◽  
Mendel Friedman
Keyword(s):  
Amino Acid ◽  
Bond Cleavage ◽  
Peptide Bond ◽  
Amino Acid Residues ◽  
Peptide Bond Cleavage

Acid Hydrolysis of Proteins for Chromatographic Analysis of Amino Acids

1987 ◽  
Vol 70 (1) ◽  
pp. 147-151 ◽  
Author(s):  
Robert W Zumwalt ◽  
Joseph S Absheer ◽  
Floyd E Kaiser ◽  
Charles W Gehrke
Keyword(s):  
Amino Acid ◽  
Matrix Effects ◽  
Bond Cleavage ◽  
Peptide Bond ◽  
Extensive Study ◽  
Performic Acid ◽  
Acid Oxidation ◽  
Peptide Bond Cleavage ◽  
Wide Range ◽  
Hydrolysis Of

Abstract The conditions used to hydrolyze proteins are vital in determining amino acid compositions because they necessarily represent a compromise aimed at yielding the best estimate of amino acid composition. Variations in ease of peptide bond cleavage, differences in amino acid stabilities, and matrix effects from nonproteinaceous components all militate against a single set of hydrolysis conditions that quantitatively hydrolyze every peptide bond and concurrently cause no destruction of any amino acid. This presentation summarizes and reviews an extensive study which evaluated a number of variations in the techniques and procedures of the classical 6N HC1, 110°C, 24 h hydrolysis of protein. The objectives of the recent investigation were: (/) to compare hydrolysis at 145°C, 4 h with 110°C, 24 h for proteins in a wide range of different sample matrixes; (2) to compare protein hydrolysis at 110°C, 24 h conducted in sealed glass ampoules after vacuum removal of air with hydrolysis in glass tubes with Teflon-lined screw caps after removal of air by vacuum, nitrogen purge, and sonication; (3) to evaluate a performic acid oxidation procedure before hydrolysis for the analysis of cystine and methionine in the different sample matrixes; (4) to evaluate multiple hydrolysis times at 145°C; (5) to evaluate the variation of interlaboratory hydrolysates prepared at 145°C, 4 h in 2 different laboratories on the amino acid analysis of an array of protein-containing matrixes. The major sources of inaccuracy and lack of precision arising from the application of ion-exchange or gas chromatography, both of which provide excellent accuracy and precision, are prechromatographic sample handling and the method used for hydrolysis of the protein sample itself. Hydrolysate preparation is the area that requires the most attention to solve problems of variability of amino acid analyses.

Development of a Reduction-Responsive Amino Acid that Induces Peptide Bond Cleavage in Hypoxic Cells

ChemBioChem ◽  
2012 ◽  
Vol 13 (7) ◽  
pp. 968-971 ◽  
Author(s):  
Akira Shigenaga ◽  
Keiji Ogura ◽  
Hiroko Hirakawa ◽  
Jun Yamamoto ◽  
Koji Ebisuno ◽  
...  
Keyword(s):  
Amino Acid ◽  
Bond Cleavage ◽  
Peptide Bond ◽  
Peptide Bond Cleavage

scholarly journals Design and synthesis of a hydrogen peroxide-responsive amino acid that induces peptide bond cleavage after exposure to hydrogen peroxide

2015 ◽  
Vol 56 (28) ◽  
pp. 4228-4231 ◽  
Author(s):  
Miku Kita ◽  
Jun Yamamoto ◽  
Takuya Morisaki ◽  
Chiaki Komiya ◽  
Tsubasa Inokuma ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Amino Acid ◽  
Bond Cleavage ◽  
Peptide Bond ◽  
Peptide Bond Cleavage ◽  
Design And Synthesis

scholarly journals Specific proteolytic modification of creatine kinase isoenzymes. Implication of C-terminal involvement in enzymic activity but not in subunit-subunit recognition

1986 ◽  
Vol 233 (1) ◽  
pp. 51-56 ◽  
Author(s):  
H G Lebherz ◽  
T Burke ◽  
J E Shackelford ◽  
J E Strickler ◽  
K J Wilson
Keyword(s):  
Creatine Kinase ◽  
Amino Acid ◽  
Bond Cleavage ◽  
Peptide Bond ◽  
Cysteine Residue ◽  
Enzymic Activity ◽  
Proteinase K ◽  
Peptide Bond Cleavage ◽  
Active Site Cysteine

We are using the isoenzymes of creatine kinase (CK) to investigate the effect of specific proteolytic modification on the abilities of enzyme subunits to establish precise subunit-subunit recognition in vitro. Previous work by others has shown that treatment of the MM isoenzyme of rabbit CK with Proteinase K results in a specific proteolytic modification and inactivation of the enzyme. In the present work, we show that both the MM and BB isoenzymes of chicken CK are also specifically modified by Proteinase K, resulting in over 98% loss of catalytic activity and approx. 10% decreases in subunit molecular masses of the enzymes. Similar reactions appear to occur when the isoenzymes are treated with Pronase E. Limited amino acid sequence analysis of intact and Proteinase K-modified MM-CK suggests that the proteolytic modification results from a single peptide-bond cleavage occurring between alanine residues 328 and 329, about 50 amino acid residues from the C-terminal end; the active-site cysteine residue was recovered in the large protein fragment of modified M-CK subunits. Proteolytically modified M-CK and B-CK subunits were able to refold and reassociate into dimeric structures after treatment with high concentrations of LiCl and at low pH. Thus the proteolytically modified CK subunits retain their ability to refold and to establish precise subunit-subunit recognition in vitro.

Cofactor processing in galactose oxidase

2003 ◽  
Vol 31 (3) ◽  
pp. 506-509 ◽  
Author(s):  
S.J. Firbank ◽  
M. Rogers ◽  
R. Hurtado-Guerrero ◽  
D.M. Dooley ◽  
M.A. Halcrow ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Bond Cleavage ◽  
Structural Similarity ◽  
Peptide Bond ◽  
Galactose Oxidase ◽  
Copper Binding ◽  
Active Site Residues ◽  
Peptide Bond Cleavage

Galactose oxidase (GO; EC 1.1.3.9) is a monomeric 68 kDa enzyme that contains a single copper and an amino acid-derived cofactor. The mechanism of this radical enzyme has been widely studied by structural, spectroscopic, kinetic and mutational approaches and there is a reasonable understanding of the catalytic mechanism and activation by oxidation to generate the radical cofactor that resides on Tyr-272, one of the copper ligands. Biogenesis of this cofactor involves the post-translational, autocatalytic formation of a thioether cross-link between the active-site residues Cys-228 and Tyr-272. This process is closely linked to a peptide bond cleavage event that releases the N-terminal 17-amino-acid pro-peptide. We have shown using pro-enzyme purified in copper-free conditions that mature oxidized GO can be formed by an autocatalytic process upon addition of copper and oxygen. Structural comparison of pro-GO (GO with the prosequence present) with mature GO reveals overall structural similarity, but with some regions showing significant local differences in main chain position and some active-site-residue side chains differing significantly from their mature enzyme positions. These structural effects of the pro-peptide suggest that it may act as an intramolecular chaperone to provide an open active-site structure conducive to copper binding and chemistry associated with cofactor formation. Various models can be proposed to account for the formation of the thioether bond and oxidation to the radical state; however, the mechanism of prosequence cleavage remains unclear.

scholarly journals Serine protease dynamics revealed by NMR analysis of the thrombin-thrombomodulin complex

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Riley B. Peacock ◽  
Taylor McGrann ◽  
Marco Tonelli ◽  
Elizabeth A. Komives
Keyword(s):  
Active Site ◽  
Serine Proteases ◽  
Protein C ◽  
Catalytic Mechanism ◽  
Bond Cleavage ◽  
Peptide Bond ◽  
Peptide Bond Cleavage ◽  
Exchange Mass Spectrometry ◽  
Catalytically Active ◽  
Hydrogen Deuterium

AbstractSerine proteases catalyze a multi-step covalent catalytic mechanism of peptide bond cleavage. It has long been assumed that serine proteases including thrombin carry-out catalysis without significant conformational rearrangement of their stable two-β-barrel structure. We present nuclear magnetic resonance (NMR) and hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments on the thrombin-thrombomodulin (TM) complex. Thrombin promotes procoagulative fibrinogen cleavage when fibrinogen engages both the anion binding exosite 1 (ABE1) and the active site. It is thought that TM promotes cleavage of protein C by engaging ABE1 in a similar manner as fibrinogen. Thus, the thrombin-TM complex may represent the catalytically active, ABE1-engaged thrombin. Compared to apo- and active site inhibited-thrombin, we show that thrombin-TM has reduced μs-ms dynamics in the substrate binding (S1) pocket consistent with its known acceleration of protein C binding. Thrombin-TM has increased μs-ms dynamics in a β-strand connecting the TM binding site to the catalytic aspartate. Finally, thrombin-TM had doublet peaks indicative of dynamics that are slow on the NMR timescale in residues along the interface between the two β-barrels. Such dynamics may be responsible for facilitating the N-terminal product release and water molecule entry that are required for hydrolysis of the acyl-enzyme intermediate.

Activation of Bovine Factor V by Thrombin and A Protease From Russell's Viper Venom (RVV)

1979 ◽  
Author(s):  
M.J. Lindhout ◽  
C. M. Jackson
Keyword(s):  
Bond Cleavage ◽  
Peptide Bond ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor V ◽  
Peptide Bond Cleavage ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Activation Products ◽  
Ca Oxalate

In order to understand the function of activated factor V in the prothrombinase complex, we isolated the activation products obtained by action of thrombin and RVV-V on factor V and studied their functional properties. Factor V isolated from plasma by means of ion-exchange chromatography, a Ca-oxalate adsorption step and gelfiltration was homogenous in SDS-gelelectrophoresis (apparent MW 360,000, with and without reduction). Increase in factor V activity upon action by RVV-V is correlated with a single peptide bond cleavage, resulting in a 270,000 dalton and a 80,000 dalton component. Additional proteolysis of factor Va(RVV/V)’ by thrombin results in a further cleavage of the high MW component into peptides with MW's of 72,000, 94,000 and about 150,000 without a furth~r increase in factor V activity. Whereas none of the isolated peptides reveal factor Va activity, activity would be generated by a recombination in the presence of Ca2+ of the 94,000 MW or 270,000 MW component with the 80,000 component. Action of thrombin alone on factor V results in peptides of MW 72,000, 80,000, 94,000 and a peptide very rich in carbohydrate with an apparent MW of 150,000.

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