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.

Cofactor processing in galactose oxidase

2004 ◽  
Vol 71 ◽  
pp. 15-25 ◽  
Author(s):  
Susan Firbank ◽  
Melanie Rogers ◽  
Ramon Hurtado Guerrero ◽  
David M. Dooley ◽  
Malcolm A. Halcrow ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Copper Ion ◽  
Extracellular Enzyme ◽  
Structural Similarity ◽  
Galactose Oxidase ◽  
Dependent Manner ◽  
Copper Binding ◽  
Active Site Residues

GO (galactose oxidase; E.C. 1.1.3.9) is a monomeric 68 kDa enzyme that contains a single copper ion and an amino acid-derived cofactor. The enzyme is produced by the filamentous fungus Fusarium graminearum as an extracellular enzyme. The enzyme has been extensively studied by structural, spectroscopic, kinetic and mutational approaches that have provided insight into the catalytic mechanism of this radical enzyme. One of the most intriguing features of the enzyme is the post-translational generation of an organic cofactor from active-site amino acid residues. Biogenesis of this cofactor involves the autocatalytic formation of a thioether bond between Cys-228 and Tyr-272, the latter being one of the copper ligands. Formation of this active-site feature is closely linked to the loss of an N-terminal 17 amino acid prosequence. When copper and oxygen are added to this pro-form of GO (pro GO), purified in copper-free conditions from the heterologous host Aspergillus nidulans, mature GO is formed by an autocatalytic process. Structural comparison of pro GO with mature GO reveals overall structural similarity, but with some regions showing significant local differences in main-chain position. Some side chains of the active-site residues differ significantly from their positions in the mature enzyme. These structural effects of the prosequence 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. The prosequence is not mandatory for processing, as a recombinant form of GO lacking this region and purified under copper-free conditions can also be processed in an autocatalytic copper- and oxygen-dependent manner.

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.

Mechanism Of Proteinase-Binding To α2-Macroglobulin

1981 ◽  
Author(s):  
L Sottrup-Jensen ◽  
H F Hansen ◽  
S B Mortensen ◽  
T E Petersen ◽  
S Magnusson ◽  
...  
Keyword(s):  
Complex Formation ◽  
Active Site ◽  
Bond Cleavage ◽  
Peptide Bond ◽  
Heat Inactivation ◽  
Complement Factor ◽  
Thiol Ester ◽  
Peptide Bond Cleavage ◽  
Bait Region ◽  
Complement Factor C3

Peptide-bond cleavage in the bait-region of the α2M polypeptide chains, associated with proteinase-α2M complex formation was shown to occur in the sequence: GLRVGFYESDVMGRG HARLVH (part of the 298-residue “middle” segment) such that plasmin, thrombin and trypsin cleave the Arg-Leu (RL) bond, elastase mainly the Val-Gly (VG) and partly the Gly-Phe (GF) bonds. Thus, in the first step of complex formation the proteinase active site binds to the bait region of α2M.J.B. Howard found that heat inactivation of α2M causes cleavage of a Glu-Glx bond, and inactivation with CH3NH2 leads to incorporation of CH3NH2 on the γ-carboxyl of the same Glx-residue. We have found that reaction of α2M with trypsin or elastase but not trypsinogen causes the 2:1 stoichiometric appearance of up to 4 SH-/tetrameric α2M for up to 2 trypsin/2 elastase bound. The -SH was found to be the Cys-SH of thiol-ester-containing PYGCGEZNM sequence. Inactivation of native α2M (by CH3NH2, heating, dissolving in 2 M guanidine or 1.6% SDS at 50°C) also led to the appearance of up to 4 SH for 4 CH3NH2 incorporated per tet- rameric α2M. Competition between trypsin and CH3NH2 for the thiol-ester site proved that trypsin binds to the same Glx- γ-carboxyl that incorporates CH3NH2. Thus, the second step of complex formation involves the thiol-ester site γ-carboxyl of α2M and a proteinase-site other than its active- site (since the complex is active against small substrates). Other compounds with nucleophiles, e.g. putrescine or insulin, when added with trypsin, were also incorporated into α2M. The thiol-ester mechanism may be associated with the rapid elimination of proteinase α2M-complexes, particularly since the recent finding by B.F. Tack that complement factor C3 has an identical thiol-ester-containing sequence, points to at least one common function of these proteins.

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.

scholarly journals High-resolution structure of the M14-type cytosolic carboxypeptidase fromBurkholderia cenocepaciarefined exploitingPDB_REDOstrategies

2014 ◽  
Vol 70 (2) ◽  
pp. 279-289 ◽  
Author(s):  
Vadim Rimsa ◽  
Thomas C. Eadsforth ◽  
Robbie P. Joosten ◽  
William N. Hunter
Keyword(s):  
Active Site ◽  
Bond Cleavage ◽  
Peptide Bond ◽  
Burkholderia Cenocepacia ◽  
Carboxy Terminus ◽  
Peptide Bond Cleavage ◽  
Terminal Domain ◽  
High Resolution Structure ◽  
Potential Substrate ◽  
Resolution Structure

A potential cytosolic metallocarboxypeptidase fromBurkholderia cenocepaciahas been crystallized and a synchrotron-radiation microfocus beamline allowed the acquisition of diffraction data to 1.9 Å resolution. The asymmetric unit comprises a tetramer containing over 1500 amino acids, and the high-throughput automated protocols embedded inPDB_REDOwere coupled with model–map inspections in refinement. This approach has highlighted the value of such protocols for efficient analyses. The subunit is constructed from two domains. The N-terminal domain has previously only been observed in cytosolic carboxypeptidase (CCP) proteins. The C-terminal domain, which carries the Zn2+-containing active site, serves to classify this protein as a member of the M14D subfamily of carboxypeptidases. Although eukaryotic CCPs possess deglutamylase activity and are implicated in processing modified tubulin, the function and substrates of the bacterial family members remain unknown. TheB. cenocepaciaprotein did not display deglutamylase activity towards a furylacryloyl glutamate derivative, a potential substrate. Residues previously shown to coordinate the divalent cation and that contribute to peptide-bond cleavage in related enzymes such as bovine carboxypeptidase are conserved. The location of a conserved basic patch in the active site adjacent to the catalytic Zn2+, where an acetate ion is identified, suggests recognition of the carboxy-terminus in a similar fashion to other carboxypeptidases. However, there are significant differences that indicate the recognition of substrates with different properties. Of note is the presence of a lysine in the S1′ recognition subsite that suggests specificity towards an acidic substrate.

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 Kringles of substrate plasminogen provide a ‘catalytic switch' in plasminogen to plasmin turnover by Streptokinase

2020 ◽  
Vol 477 (5) ◽  
pp. 953-970
Author(s):  
Vandna Sharma ◽  
Shekhar Kumar ◽  
Girish Sahni
Keyword(s):  
Chemical Transformation ◽  
Active Site ◽  
Catalytic Domain ◽  
Bond Cleavage ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Peptide Bond ◽  
Peptide Bond Cleavage ◽  
Catalytic Processing ◽  
Kinetics Of

To understand the role of substrate plasminogen kringles in its differential catalytic processing by the streptokinase — human plasmin (SK-HPN) activator enzyme, Fluorescence Resonance Energy Transfer (FRET) model was generated between the donor labeled activator enzyme and the acceptor labeled substrate plasminogen (for both kringle rich Lys plasminogen — LysPG, and kringle less microplasminogen — µPG as substrates). Different steps of plasminogen to plasmin catalysis i.e. substrate plasminogen docking to scissile peptide bond cleavage, chemical transformation into proteolytically active product, and the decoupling of the nascent product from the SK-HPN activator enzyme were segregated selectively using (1) FRET signal as a proximity sensor to score the interactions between the substrate and the activator during the cycle of catalysis, (2) active site titration studies and (3) kinetics of peptide bond cleavage in the substrate. Remarkably, active site titration studies and the kinetics of peptide bond cleavage have shown that post docking chemical transformation of the substrate into the product is independent of kringles adjacent to the catalytic domain (CD). Stopped-flow based rapid mixing experiments for kringle rich and kringle less substrate plasminogen derivatives under substrate saturating and single cycle turnover conditions have shown that the presence of kringle domains adjacent to the CD in the macromolecular substrate contributes by selectively speeding up the final step, namely the product release/expulsion step of catalysis by the streptokinase-plasmin(ogen) activator enzyme.

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