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.

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.

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.

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.

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.

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.

scholarly journals Short hydrogen bonds in the catalytic mechanism of serine proteases

2008 ◽  
Vol 73 (4) ◽  
pp. 393-403 ◽  
Author(s):  
Vladimir Leskovac ◽  
Svetlana Trivic ◽  
Draginja Pericin ◽  
Mira Popovic ◽  
Julijan Kandrac
Keyword(s):  
Hydrogen Bond ◽  
Hydrogen Bonds ◽  
Active Site ◽  
Serine Proteases ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Data Bank ◽  
Ground State Structure ◽  
Low Barrier Hydrogen Bonds ◽  
Short Hydrogen Bonds

The survey of crystallographic data from the Protein Data Bank for 37 structures of trypsin and other serine proteases at a resolution of 0.78-1.28 ? revealed the presence of hydrogen bonds in the active site of the enzymes, which are formed between the catalytic histidine and aspartate residues and are on average 2.7 ? long. This is the typical bond length for normal hydrogen bonds. The geometric properties of the hydrogen bonds in the active site indicate that the H atom is not centered between the heteroatoms of the catalytic histidine and aspartate residues in the active site. Taken together, these findings exclude the possibility that short "low-barrier" hydrogen bonds are formed in the ground state structure of the active sites examined in this work. Some time ago, it was suggested by Cleland that the "low-barrier hydrogen bond" hypothesis is operative in the catalytic mechanism of serine proteases, and requires the presence of short hydrogen bonds around 2.4 ? long in the active site, with the H atom centered between the catalytic heteroatoms. The conclusions drawn from this work do not exclude the validity of the "low-barrier hydrogen bond" hypothesis at all, but they merely do not support it in this particular case, with this particular class of enzymes.

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