scholarly journals Mechanism of activation of the gastric aspartic proteinases: pepsinogen, progastricsin and prochymosin

1998 ◽  
Vol 335 (3) ◽  
pp. 481-490 ◽  
Author(s):  
Catherine RICHTER ◽  
Takuji TANAKA ◽  
Rickey Y. YADA
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Electrostatic Interactions ◽  
Bond Cleavage ◽  
Active Form ◽  
Active Enzyme ◽  
Aspartic Proteinases ◽  
Active Centre ◽  
Activation Reaction ◽  
Molecular Reaction

The gastric aspartic proteinases (pepsin A, pepsin B, gastricsin and chymosin) are synthesized in the gastric mucosa as inactive precursors, known as zymogens. The gastric zymogens each contain a prosegment (i.e. additional residues at the N-terminus of the active enzyme) that serves to stabilize the inactive form and prevent entry of the substrate to the active site. Upon ingestion of food, each of the zymogens is released into the gastric lumen and undergoes conversion into active enzyme in the acidic gastric juice. This activation reaction is initiated by the disruption of electrostatic interactions between the prosegment and the active enzyme moiety at acidic pH values. The conversion of the zymogen into its active form is a complex process, involving a series of conformational changes and bond cleavage steps that lead to the unveiling of the active site and ultimately the removal and dissociation of the prosegment from the active centre of the enzyme. During this activation reaction, both the prosegment and the active enzyme undergo changes in conformation, and the proteolytic cleavage of the prosegment can occur in one or more steps by either an intra- or inter-molecular reaction. This variability in the mechanism of proteolysis appears to be attributable in part to the structure of the prosegment. Because of the differences in the activation mechanisms among the four types of gastric zymogens and between species of the same zymogen type, no single model of activation can be proposed. The mechanism of activation of the gastric aspartic proteinases and the contribution of the prosegment to this mechanism are discussed, along with future directions for research.

scholarly journals Activation and selectivity of OTUB-1 and OTUB-2 deubiquitinylases

2020 ◽  
Vol 295 (20) ◽  
pp. 6972-6982
Author(s):  
Dakshinamurthy Sivakumar ◽  
Vikash Kumar ◽  
Michael Naumann ◽  
Matthias Stein
Keyword(s):  
Active Site ◽  
Electrostatic Interactions ◽  
Active Form ◽  
Cysteine Proteases ◽  
Binding Pocket ◽  
Catalytic Triad ◽  
Catalytic Site ◽  
Dynamics Simulation ◽  
Active Site Residues ◽  
Catalytically Active

The ovarian tumor domain (OTU) deubiquitinylating cysteine proteases OTUB1 and OTUB2 (OTU ubiquitin aldehyde binding 1 and 2) are representative members of the OTU subfamily of deubiquitinylases. Deubiquitinylation critically regulates a multitude of important cellular processes, such as apoptosis, cell signaling, and growth. Moreover, elevated OTUB expression has been observed in various cancers, including glioma, endometrial cancer, ovarian cancer, and breast cancer. Here, using molecular dynamics simulation approaches, we found that both OTUB1 and OTUB2 display a catalytic triad characteristic of proteases but differ in their configuration and protonation states. The OTUB1 protein had a prearranged catalytic site, with strong electrostatic interactions between the active-site residues His265 and Asp267. In OTUB2, however, the arrangement of the catalytic triad was different. In the absence of ubiquitin, the neutral states of the catalytic-site residues in OTUB2 were more stable, resulting in larger distances between these residues. Only upon ubiquitin binding did the catalytic triad in OTUB2 rearrange and bring the active site into a catalytically feasible state. An analysis of water access channels revealed only a few diffusion trajectories for the catalytically active form of OTUB1, whereas in OTUB2 the catalytic site was solvent-accessible, and a larger number of water molecules reached and left the binding pocket. Interestingly, in OTUB2, the catalytic residues His224 and Asn226 formed a stable hydrogen bond. We propose that the observed differences in activation kinetics, protonation states, water channels, and active-site accessibility between OTUB1 and OTUB2 may be relevant for the selective design of OTU inhibitors.

scholarly journals Aldolase A Ins(1,4,5)P3-binding domains as determined by site-directed mutagenesis

1999 ◽  
Vol 341 (3) ◽  
pp. 805-812 ◽  
Author(s):  
Carl B. BARON ◽  
Dean R. TOLAN ◽  
Kyung H. CHOI ◽  
Ronald F. COBURN
Keyword(s):  
Catalytic Activity ◽  
Active Site ◽  
Electrostatic Interactions ◽  
Bond Cleavage ◽  
Site Directed Mutagenesis ◽  
Binding Domains ◽  
Carbon Carbon Bond ◽  
Catalytically Active ◽  
Aldolase Activity ◽  
Aldolase A

We substituted neutral amino acids for some positively charged residues (R42, K107, K146, R148 and K229) that line the active site of aldolase A in an effort to determine binding sites for inositol 1,4,5-trisphosphate. In addition, D33 (involved in carbon-carbon bond cleavage) was mutated. K229A and D33S aldolases showed almost no catalytic activity, but Ins(1,4,5)P3 binding was similar to that determined with the use of wild-type aldolase A. R42A, K107A, K146R and R148A had markedly decreased affinities for Ins(1,4,5)P3 binding, increased EC50 values for Fru(1,6)P2-evoked release of bound Ins(1,4,5)P3 and increased Ki values for Ins(1,4,5)P3-evoked inhibition of aldolase activity. K146Q (positive charge removal) had essentially no catalytic activity and could not bind Ins(1,4,5)P3. Computer-simulated docking of Ins(1,4,5)P3 in the aldolase A structure was consistent with electrostatic binding of Ins(1,4,5)P3 to K107, K146, R148, R42, R303 and backbone nitrogens, as has been reported for Fru(1,6)P2 binding. Results indicate that Ins(1,4,5)P3 binding occurs at the active site and is not dependent on having a catalytically active enzyme; they also suggest that there is competition between Ins(1,4,5)P3 and Fru(1,6)P2 for binding. Although Ins(1,4,5)P3 binding to aldolase involved electrostatic interactions, the aldolase A Ins(1,4,5)P3-binding domain did not show other similarities to pleckstrin homology domains or phosphotyrosine-binding domains known to bind Ins(1,4,5)P3 in other proteins.

scholarly journals Electrostatic interactions affecting the active site of class Sigma glutathione S-transferase

2000 ◽  
Vol 347 (1) ◽  
pp. 193-197 ◽  
Author(s):  
Julie M. STEVENS ◽  
Richard N. ARMSTRONG ◽  
Heini W. DIRR
Keyword(s):  
Ionic Strength ◽  
Conformational Changes ◽  
Active Site ◽  
Electrostatic Interactions ◽  
Size Exclusion ◽  
Tryptophan Residue ◽  
Glutathione S Transferase ◽  
Protein Fluorescence ◽  
Subunit Interface ◽  
Α Helix

We have shown previously that the solvent-induced equilibrium unfolding mechanism of class Sigma glutathione S-transferase (GST) is strongly affected by ionic strength [Stevens, Hornby, Armstrong and Dirr (1998) Biochemistry 37, 15534-15541]. The protein is dimeric and has a hydrophilic subunit interface. Here we show that ionic strength alone has significant effects on the conformation of the protein, in particular at the active site. With the use of NaCl at up to 2 M under equilibrium conditions, the protein lost 60% of its catalytic activity and the single tryptophan residue per subunit became partly exposed. The effect was independent of protein concentration, eliminating the dissociation of the dimer as a possibility for the conformational changes. This was confirmed by size-exclusion HPLC. There was no significant change in the secondary structure of the protein according to far-UV CD data. Manual-mixing and stopped-flow kinetics experiments showed a slow single-exponential salt-induced change in protein fluorescence. For equilibrium and kinetics experiments, the addition of an active-site ligand (S-hexylglutathione) completely protected the protein from the ionic-strength-induced conformational changes. This suggests that the change occurs at or near the active site. Possible structural reasons for these novel effects are proposed, such as the flexibility of the α-helix 2 region as well as the hydrophilic subunit interface, highlighting the importance of electrostatic interactions in maintaining the structure of the active site of this GST.

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.

scholarly journals Active site lysine backbone undergoes conformational changes in the bacteriorhodopsin photocycle.

1994 ◽  
Vol 269 (10) ◽  
pp. 7387-7389
Author(s):  
H. Takei ◽  
Y. Gat ◽  
Z. Rothman ◽  
A. Lewis ◽  
M. Sheves
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Bacteriorhodopsin Photocycle

Specific protein-membrane interactions promote packaging of metallo-β-lactamases into outer membrane vesicles

2021 ◽  
Author(s):  
Carolina López ◽  
Alessio Prunotto ◽  
Guillermo Bahr ◽  
Robert A. Bonomo ◽  
Lisandro J. González ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Electrostatic Interactions ◽  
Active Form ◽  
Membrane Vesicles ◽  
Specific Protein ◽  
Outer Membrane Vesicles ◽  
Soluble Enzyme ◽  
Membrane Interactions ◽  
Resistance Determinants ◽  
Protein Membrane

Outer membrane vesicles (OMVs) act as carriers of bacterial products such as plasmids and resistance determinants, including metallo-β-lactamases. The lipidated, membrane-anchored metallo-β-lactamase NDM-1 can be detected in Gram-negative OMVs. The soluble domain of NDM-1 also forms electrostatic interactions with the membrane. Herein, we show that these interactions promote its packaging into OMVs produced by Escherichia coli . We report that favorable electrostatic protein-membrane interactions are also at work in the soluble enzyme IMP-1, while being absent in VIM-2. These interactions correlate with an enhanced incorporation of IMP-1 compared to VIM-2 into OMVs. Disruption of these interactions in NDM-1 and IMP-1 impairs their inclusion into vesicles, confirming their role in defining the protein cargo in OMVs. These results also indicate that packaging of metallo-β-lactamases into vesicles in their active form is a common phenomenon that involves cargo selection based on specific molecular interactions.

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