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Interleukins and Metalloproteinases in Arthritis
Published in Thomas F. Kresina, Monoclonal Antibodies, Cytokines, and Arthritis, 2020
The design of the inhibitors was based on the premise that proteoglycanase and collagenase interactions with their substrates were similar to those of the zinc-dependent endoproteinase, thermolysin. Also considered were the analyses of the cleavage products of several small peptide substrates and the inhibitor profiles of thermolysin and proteoglycanase (86).
Proteases as Biocatalysts for the Synthesis of Model Peptides
Published in Willi Kullmann, Enzymatic Peptide Synthesis, 1987
Thermolysin, a neutral metalloprotease isolated from Bacillus thermoproteolyticus has frequently been used in enzymatic peptide synthesis. The enzyme binds one zinc ion essential for catalytic activity,95 and four calcium ions, which are required for optimal thermostability.96Thermolysin exhibits a strong preference for peptide bonds the imino group of which is contributed by bulky hydrophobic amino acid residues.55 In addition, the enzymatic activity is further enhanced when the carbonyl portion of the sensitive bond is donated by a hydrophobic residue.97 Thus, thermolysin represents a rare case, where the primary specificity of a protease is predominantly determined by structural features of the -site of the substrates. Current information on the mechanism of thermolysin-action is rather contradictory. A general base catalysis has been postulated.98 Alternatively, catalytic pathways involving both an acyl-enzyme complex — via an anhydride linkage — and an amino-acyl complex are favored by other authors.99
The Modification of Histidine Residues
Published in Roger L. Lundblad, Chemical Reagents for Protein Modification, 2020
Neurath and co-workers39 have examined the reaction of thermolysin with diethylpyrocarbonate (ethoxyformic anhydride) in some detail. The time course for the loss of catalytic activity by thermolysin upon reaction with diethylpyrocarbonate is shown in Figure 8. Also shown in Figure 8 is the recovery of activity upon reaction of the modified protein with hydroxylamine. Under these reaction conditions 13.4 carboethoxy groups were incorporated per mole of enzyme. The presence of carbobenzoxy-L-phenylalanine, a competitive inhibitor, protected the enzyme from inactivation and reduced the extent of modification to 12.5 mol/mol protein. It should be noted that the reaction demonstrated a pH dependence (Figure 9) consistent with the modification of histidine. Figure 10 shows the difference spectra of thermolysin (panel A) after reaction with diethylpyrocarbonate (solid line) and then after subsequent reaction with 0.020 M neutral hydroxylamine (broken line). This is compared with panel B where the difference spectrum obtained on the reaction of diethylpyrocarbonate with N-acetyl-L-tyrosine ethyl ester (solid line) or imidazole (broken line); the dotted line is the algebraic sum of the two reactions. The increase at 242 nm is consistent with the modification of histidine while the decrease at 278 to 280 nm is indicative of tyrosine modification. The tyrosyl residues modified during the reaction were not regenerated on reaction with 0.020 M hydroxylamine (Figure 11). These investigators also make the point that there is a significant decrease in the absorbance of N-acetyl-L-tyrosine ethyl ester at 234 nm (Figure 10, panel B) which would affect the accuracy of measurement at 240 to 242 nm for the determination of the extent of histidine modification. These investigators found it more accurate to determine the extent of histidine modification by spectral measurement during hydroxylamine reactivation.
Correlation of Staphylococcus Epidermidis Phenotype and Its Corneal Virulence
Published in Current Eye Research, 2021
Armando R. Caballero, Aihua Tang, Michael Bierdeman, Richard O’Callaghan, Mary Marquart
E coli M15 [pREP4] expressing the recombinant protease was grown in LB media to an OD600 ~ 0.6 and induced with 0.5 mM IPTG overnight. Afterwards, the bacteria were pelleted and the culture supernatant was filtered through a 0.45 µm filter, concentrated and dialyzed. The soluble recombinant protein was purified by affinity chromatography under non-denaturing conditions (TALON Metal Affinity Resin [Clontech Laboratories, Mountain View, CA]) following the manufacturer’s instructions and concentrated. Aliquots of the purified rEsp (200 µl) were incubated with 4 µg of thermolysin from Geobacillus stearothermophilus (Sigma-Aldrich, St. Louis, MO) at 37ºC for 30 minutes to cleave the pro-peptide and the reaction was terminated with EDTA. The activated rEsp was purified away from thermolysin by molecular sieve chromatography.
Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine, two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeutics
Published in mAbs, 2019
Oksana Tyshchuk, Christoph Gstöttner, Dennis Funk, Simone Nicolardi, Stefan Frost, Stefan Klostermann, Tim Becker, Elena Jolkver, Felix Schumacher, Claudia Ferrara Koller, Hans Rainer Völger, Manfred Wuhrer, Patrick Bulau, Michael Mølhøj
The antibodies were denatured and reduced in 0.3 M Tris-HCl pH 8, 6 M guanidine-HCl and 20 mM dithiothreitol (DTT) at 37°C for 1 h, and alkylated by adding 40 mM iodoacetic acid (C13: 99%) (Sigma-Aldrich) at room temperature in the dark for 15 min. Excess iodoacetic acid was inactivated by adding DTT to 40 mM. The alkylated fusion protein was buffer exchanged using NAP5 gel filtration columns, and a proteolytic digestion with trypsin was performed in 50 mM Tris-HCl, pH 7.5 at 37°C for 16 h. The reaction was stopped by adding formic acid to 0.4% (v/v). Thermolysin digests were performed as described by Tyshchuk et al.11 Digested samples were stored at −80°C and analyzed by UPLC-MS/MS using a nanoAcquity UPLC (Waters) and an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). About 2.5 µg digested fusion protein was injected in 5 µL. Chromatographic separation was performed by reversed-phase on a BEH300 C18 column (1 × 150 mm, 1.7 µm) or a CSH130 C18 column, 1 × 150 mm, 1.7 µm (Waters) using mobile phase A and B containing 0.1% (v/v) formic acid in UPLC grade water and acetonitrile, respectively, 60 µL/min flow rate, 50°C column temperature, and the following gradient: 1% mobile phase B [0–3 min], 1% to 40% mobile phase B [3–93 min], 40% to 99% mobile phase B [93–94 min], 99% mobile phase B [94–96 min], 99% to 1% mobile phase B [96–97 min], and 1% mobile phase B [97–105 min]. Two injections of mobile phase A were performed between sample injections using a similar 50 min gradient up to 99% mobile phase B to prevent carry-over between samples. Synthetic peptides were spiked into to digests at different levels.
Inhibition of bacterial and human zinc-metalloproteases by bisphosphonate- and catechol-containing compounds
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2021
Fatema Rahman, Tra-Mi Nguyen, Olayiwola A. Adekoya, Cristina Campestre, Paolo Tortorella, Ingebrigt Sylte, Jan-Olof Winberg
TLN from Bacillus thermoproteolyticus is the model enzyme of the M4 family of proteases, which is also termed the thermolysin family50. These enzymes have a zinc ion in the catalytic site, which has tetrahedral coordination. Two histidines of a HEXXH motif and a glutamic acid located 18–72 residues C-terminal to the HEXXH motif are the three ligands that anchor the zinc ion to the enzyme, while the fourth ligand is a water molecule as in the MMPs, which also binds the side chain of the glutamate following the first histidine in the zinc binding segment8,9,50. Inhibitors containing a metal binding group replace the catalytic water molecule on the zinc ion when they bind the catalytic site51. TLN, PLN from Pseudomonas aeruginosa (LasB or elastase of P. aeruginosa) and aureolysin (ALN) from Staphylococcus aureus belong to the subclan MA(E) of the M4 family, also known as the “Glu-zincins”8,9,50. These three proteases have several similarities despite a modest sequence identity (28% between TLN and PLN)52,53. The three dimensional (3D) structures of PLN and TLN have been extensively studied, also in complex with inhibitors, and reveal large similarities in the overall structure. The main structural differences are that PLN consists of a slightly more open substrate binding cleft than TLN, and that PLN has one structural calcium while TLN has three53–55. For ALN only the 3 D-structure of the free enzyme is known56. Although PLN is not as well characterised as TLN, it appears that the slight difference in substrate specificity between the two enzymes is mainly due to the size of the S1′-subpocket and a more open substrate binding cleft in PLN than in TLN. PLN has a broader substrate specificity than most other M4 family members including TLN, although all these enzymes prefer a hydrophobic amino acid at the P1’ position. Furthermore, for substrate degradation four subsites of PLN require to be occupied50,53,55.