scholarly journals Role of lysine, tryptophan and calcium in the β-elimination activity of a low-molecular-mass pectate lyase from Fusarium moniliformae

1996 ◽  
Vol 319 (1) ◽  
pp. 159-164 ◽  
Author(s):  
M. Narsimha RAO ◽  
Asha A. KEMBHAVI ◽  
Aditi PANT
Keyword(s):  
Fluorescence Quenching ◽  
Molecular Mass ◽  
Active Site ◽  
Structural Changes ◽  
Substrate Binding ◽  
Gel Filtration ◽  
Pectate Lyase ◽  
Tryptophan Fluorescence ◽  
Emission Spectrometry ◽  
Substrate Affinity

An extracellular pectate lyase from Fusarium moniliformae was purified to homogeneity by affinity chromatography followed by gel filtration, with a yield of 76.5%. Laser desorption MS of the enzyme gave a molecular mass of 12133.5±2.5 Da. The pectate lyase was a glycoprotein with a 5% carbohydrate content and had a pI value of 9.1. Atomic-emission spectrometry showed that Ca2+ was a part of the holoenzyme held by carboxy groups of the protein. These results support the hypothesis of a putative Ca2+ site suggested by Yodder, Keen and Jurnak [(1993) Science 260, 1503–1507] in the crystal structure of pectate lyase C of Erwinia chrysanthemi. Loss of Ca2+ was observed by treatment with EGTA or carboxy-modifying Woodward's reagent K, with subsequent loss of enzyme activity. Tryptophan fluorescence quenching showed that Ca2+ does not affect binding of substrate to enzyme. Chemical-modification and substrate-protection studies showed the presence of lysine and tryptophan at or near the active site of the pectate lyase. Chemically modified enzyme showed no major structural changes as determined by CD. Amino acid analyses of native, trinitrobenzenesulphonate (TBNS)-treated and substrate-protected TNBS-treated enzyme showed that a single essential residue of lysine is present at or near the active-site. Substrate-affinity studies showed that tryptophan could be essential for substrate binding, whereas lysine could be involved in the catalysis. Fluorescence quenching further confirmed the involvement of tryptophan in substrate binding. The reaction mechanism involving β-elimination by this enzyme is discussed.

Structural and mechanistic insight into how antibodies inhibit serine proteases

2010 ◽  
Vol 430 (2) ◽  
pp. 179-189 ◽  
Author(s):  
Rajkumar Ganesan ◽  
Charles Eigenbrot ◽  
Daniel Kirchhofer
Keyword(s):  
Active Site ◽  
Serine Proteases ◽  
Structural Changes ◽  
Competitive Inhibition ◽  
Substrate Binding ◽  
Structural Features ◽  
Inhibition Mechanism ◽  
Interaction Sites ◽  
Binding Cleft ◽  
Insight Into

Antibodies display great versatility in protein interactions and have become important therapeutic agents for a variety of human diseases. Their ability to discriminate between highly conserved sequences could be of great use for therapeutic approaches that target proteases, for which structural features are conserved among family members. Recent crystal structures of antibody–protease complexes provide exciting insight into the variety of ways antibodies can interfere with the catalytic machinery of serine proteases. The studies revealed the molecular details of two fundamental mechanisms by which antibodies inhibit catalysis of trypsin-like serine proteases, exemplified by hepatocyte growth factor activator and MT-SP1 (matriptase). Enzyme kinetics defines both mechanisms as competitive inhibition systems, yet, on the molecular level, they involve distinct structural elements of the active-site region. In the steric hindrance mechanism, the antibody binds to protruding surface loops and inserts one or two CDR (complementarity-determining region) loops into the enzyme's substrate-binding cleft, which results in obstruction of substrate access. In the allosteric inhibition mechanism the antibody binds outside the active site at the periphery of the substrate-binding cleft and, mediated through a conformational change of a surface loop, imposes structural changes at important substrate interaction sites resulting in impaired catalysis. At the centre of this allosteric mechanism is the 99-loop, which is sandwiched between the substrate and the antibody-binding sites and serves as a mobile conduit between these sites. These findings provide comprehensive structural and functional insight into the molecular versatility of antibodies for interfering with the catalytic machinery of proteases.

scholarly journals A kinase bioscavenger provides antibiotic resistance by extremely tight substrate binding

2020 ◽  
Vol 6 (26) ◽  
pp. eaaz9861 ◽  
Author(s):  
Stanislav S. Terekhov ◽  
Yuliana A. Mokrushina ◽  
Anton S. Nazarov ◽  
Alexander Zlobin ◽  
Arthur Zalevsky ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Microbial Communities ◽  
Active Site ◽  
Substrate Binding ◽  
Catalytic Efficiency ◽  
Crystallographic Analysis ◽  
Diffusion Limit ◽  
Substrate Affinity ◽  
Multiscale Simulations ◽  
Substrate Promiscuity

Microbial communities are self-controlled by repertoires of lethal agents, the antibiotics. In their turn, these antibiotics are regulated by bioscavengers that are selected in the course of evolution. Kinase-mediated phosphorylation represents one of the general strategies for the emergence of antibiotic resistance. A new subfamily of AmiN-like kinases, isolated from the Siberian bear microbiome, inactivates antibiotic amicoumacin by phosphorylation. The nanomolar substrate affinity defines AmiN as a phosphotransferase with a unique catalytic efficiency proximal to the diffusion limit. Crystallographic analysis and multiscale simulations revealed a catalytically perfect mechanism providing phosphorylation exclusively in the case of a closed active site that counteracts substrate promiscuity. AmiN kinase is a member of the previously unknown subfamily representing the first evidence of a specialized phosphotransferase bioscavenger.

scholarly journals ω-Oxidation impairs oxidizability of polyenoic fatty acids by 15-lipoxygenases: consequences for substrate orientation at the active site

1998 ◽  
Vol 336 (2) ◽  
pp. 345-352 ◽  
Author(s):  
Igor IVANOV ◽  
Kristin SCHWARZ ◽  
Herman G. HOLZHÜTTER ◽  
Galina MYAGKOVA ◽  
Hartmut KÜHN
Keyword(s):  
Fatty Acids ◽  
Active Site ◽  
Substrate Binding ◽  
Salt Bridge ◽  
Binding Pocket ◽  
Substrate Affinity ◽  
Carboxylate Group ◽  
Fatty Acid Binding ◽  
Substrate Orientation ◽  
Binding Cleft

During oxygenation by 15-lipoxygenases, polyenoic fatty acids are bound at the active site in such a way that the ω-terminus of the fatty acids penetrates into the substrate binding pocket. In contrast, for arachidonic acid 5-lipoxygenation, an inverse head to tail orientation has been suggested. However, an inverse orientation may be hindered by the large energy barrier associated with burying the charged carboxylate group in the hydrophobic environment of the substrate binding cleft. We studied the oxygenation kinetics of ω-modified fatty acids by 15-lipoxygenases and found that ω-hydroxylation strongly impaired substrate affinity (higher Km), but only moderately altered Vmax. In contrast, ω-carboxylation completely prevented the lipoxygenase reaction; however, methylation of the additional carboxylate group restored the activity. Arg403 of the human 15-lipoxygenase has been implicated in fatty acid binding by forming a salt bridge with the carboxylate group, and thus mutation of this amino acid to an uncharged residue was supposed to favour an inverse substrate orientation. The prepared Arg403 → Leu mutant of the rabbit 15-lipoxygenase was found to be a less effective catalyst of linoleic acid oxygenation. However, the oxygenation rate of ω-hydroxyarachidonic acid was similar when the wild-type and mutant enzyme were compared, and the patterns of oxygenation products were identical for both enzyme species. These data suggest that introduction of a polar, or even charged residue, at the ω-terminus of substrate fatty acids in connection with mutation of Arg403 may not alter substrate alignment at the active site of 15-lipoxygenases.

scholarly journals Tryptophan fluorescence quenching in β-lactam-interacting proteins is modulated by the structure of intermediates and final products of the acylation reaction

2018 ◽  
Author(s):  
Sébastien Triboulet ◽  
Zainab Edoo ◽  
Fabrice Compain ◽  
Clément Ourghanlian ◽  
Adrian Dupuis ◽  
...  
Keyword(s):  
Fluorescence Quenching ◽  
Active Site ◽  
Tryptophan Fluorescence ◽  
Multidrug Resistant ◽  
Enzyme Inactivation ◽  
Cross Linking ◽  
Mesomeric Effect ◽  
Resistant Tuberculosis ◽  
The Status ◽  
Covalent Adducts

In most bacteria, β-lactam antibiotics inhibit the last cross-linking step of peptidoglycan synthesis by acylation of the active-site Ser of D,D-transpeptidases belonging to the penicillin-binding protein (PBP) family. In mycobacteria, cross-linking is mainly ensured by L,D-transpeptidases (LDTs), which are promising targets for the development of β-lactam-based therapies for multidrug-resistant tuberculosis. For this purpose, fluorescence spectroscopy is used to investigate the efficacy of LDT inactivation by β-lactams but the basis for fluorescence quenching during enzyme acylation remains unknown. In contrast to what has been reported for PBPs, we show here using a model L,D-transpeptidase (Ldtfm) that fluorescence quenching of Trp residues does not depend upon direct hydrophobic interaction between Trp residues and β-lactams. Rather, Trp fluorescence was quenched by the drug covalently bound to the active-site Cys residue of Ldtfm. Fluorescence quenching was not quantitatively determined by the size of the drug and was not specific of the thioester link connecting the β-lactam carbonyl to the catalytic Cys as quenching was also observed for acylation of the active-site Ser of β-lactamase BlaC from M. tuberculosis. Fluorescence quenching was extensive for reaction intermediates containing an amine anion and for acylenzymes containing an imine stabilized by mesomeric effect, but not for acylenzymes containing a protonated β-lactam nitrogen. Together, these results indicate that the extent of fluorescence quenching is determined by the status of the β-lactam nitrogen. Thus, fluorescence kinetics can provide information not only on the efficacy of enzyme inactivation but also on the structure of the covalent adducts responsible for enzyme inactivation.

Turnover of Human Extrinsic (Tissue-Type) Plasminogen Activator in Rabbits

1981 ◽  
Vol 46 (03) ◽  
pp. 658-661 ◽  
Author(s):  
C Korninger ◽  
J M Stassen ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Intravenous Injection ◽  
Active Site ◽  
Half Life ◽  
Degradation Products ◽  
Gel Filtration ◽  
Tissue Type ◽  
Organ Distribution ◽  
Small Rise

SummaryThe turnover of highly purified human extrinsic plasminogen activator (EPA) (one- and two-chain form) was studied in rabbits. Following intravenous injection, EPA-activity declined rapidly. The disappearance rate of EPA from the plasma could adequately be described by a single exponential term with a t ½ of approximately 2 min for both the one-chain and two-chain forms of EPA.The clearance and organ distribution of EPA was studied by using 125I-labeled preparations. Following intravenous injection of 125I-1abeled EPA the radioactivity disappeared rapidly from the plasma also with a t ½ of approximately 2 min down to a level of 15 to 20 percent, followed by a small rise of blood radioactivity. Gel filtration of serial samples revealed that the secondary increase of the radioactivity was due to the reappearance of radioactive breakdown products in the blood. Measurement of the organ distribution of 125I at different time intervals revealed that EPA was rapidly accumulated in the liver, followed by a release of degradation products in the blood.Experimental hepatectomy markedly prolonged the half-life of EPA in the blood. Blocking the active site histidine of EPA had no effect on the half-life of EPA in blood nor on the gel filtration patterns of 125I in serial plasma samples.It is concluded that human EPA is rapidly removed from the blood of rabbits by clearance and degradation in the liver. Recognition by the liver does not require a functional active site in the enzyme. Neutralization in plasma by protease inhibitors does not represent a significant pathway of EPA inactivation in vivo.

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