Chemical synthesis and enzymatic properties of RNase A analogues designed to enhance second-step catalytic activity

2016 ◽  
Vol 14 (37) ◽  
pp. 8804-8814
Author(s):  
David J. Boerema ◽  
Valentina A. Tereshko ◽  
Junliang Zhang ◽  
Stephen B. H. Kent
Keyword(s):  
Catalytic Activity ◽  
Chemical Synthesis ◽  
Rnase A ◽  
Enzymatic Properties ◽  
Second Step ◽  
Enzyme Molecule ◽  
Rate Of Hydrolysis

Adenine covalently attached to the RNase A enzyme molecule decreased the rate of transphosphorylation and increased the rate of hydrolysis.

Chemical Synthesis of an Enzyme Containing an Artificial Catalytic Apparatus

2020 ◽  
Vol 73 (4) ◽  
pp. 321
Author(s):  
Vladimir Torbeev ◽  
Stephen B. H. Kent
Keyword(s):  
Catalytic Activity ◽  
Chemical Synthesis ◽  
Enzyme Catalysis ◽  
Polypeptide Chain ◽  
Peptide Bond ◽  
Enzyme Molecule ◽  
Peptide Substrates ◽  
Unique Approach ◽  
Hiv 1 ◽  
Residue Polypeptide

With the goal of investigating electronic aspects of the catalysis of peptide bond hydrolysis, an analogue of HIV-1 protease was designed in which a non-peptide hydroxy-isoquinolinone artificial catalytic apparatus replaced the conserved Asp25–Thr26–Gly27 sequence in each 99-residue polypeptide chain of the homodimeric enzyme molecule. The enzyme analogue was prepared by total chemical synthesis and had detectable catalytic activity on known HIV-1 protease peptide substrates. Compared with uncatalyzed hydrolysis, the analogue enzyme increased the rate of peptide bond hydrolysis by ∼108-fold. Extensions of this unique approach to the study of enzyme catalysis in HIV-1 protease are discussed.

ÉTUDE DES EFFETS ÉLECTROSTATIQUES SUR LE MÉCANISME D'ACTION CATALYTIQUE DE LA PHOSPHATASE ALCALINE INTESTINALE DE VEAU

1965 ◽  
Vol 43 (8) ◽  
pp. 2222-2235 ◽  
Author(s):  
Michel Lazdunski ◽  
Jacques Brouillard ◽  
Ludovic Ouellet
Keyword(s):  
Dielectric Constant ◽  
Ionic Strength ◽  
Maximum Activity ◽  
Enzyme Molecule ◽  
High Substrate Concentration ◽  
Nitrophenyl Phosphate ◽  
Phosphatase Alcaline ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Activated Complex

The influence of dioxane and ethanol on the rate of hydrolysis of p-nitrophenyl phosphate in the presence of an intestinal alcaline phosphatase can be interpreted as a dielectric constant effect, at high substrate concentration. The dielectric constant effect is a function of the pH of the medium and is maximum around pH 9.4 at 25 °C and pH 9.0 at 15 °C. An interpretation suggesting that the change in diameter of the enzyme molecule becoming an activated complex is minimum at a pH of maximum activity is proposed. The same model can take into account the influence of the ionic strength on the same reaction.

scholarly journals Endocytotic Internalization as a Crucial Factor for the Cytotoxicity of Ribonucleases

2007 ◽  
Vol 282 (38) ◽  
pp. 27640-27646 ◽  
Author(s):  
Franziska Leich ◽  
Nadine Stöhr ◽  
Anne Rietz ◽  
Renate Ulbrich-Hofmann ◽  
Ulrich Arnold
Keyword(s):  
Catalytic Activity ◽  
Cellular Uptake ◽  
Cellular Membrane ◽  
Subcellular Fractionation ◽  
Rnase A ◽  
Cytotoxic Action ◽  
Ribonuclease Inhibitor ◽  
Proteolytic Stability ◽  
K 562 Cells

The cytotoxic action of ribonucleases (RNases) requires the interaction of the enzyme with the cellular membrane, its internalization, translocation to the cytosol, and the degradation of ribonucleic acid. The interplay of these processes as well as the role of the thermodynamic and proteolytic stability, the catalytic activity, and the evasion from the intracellular ribonuclease inhibitor (RI) has not yet been fully elucidated. As cytosolic internalization is indispensable for the cytotoxicity of extracellular ribonucleases, we investigated the extent of cytosolic internalization of a cytotoxic, RI-evasive RNase A variant (G88R-RNase A) and of various similarly cytotoxic but RI-sensitive RNase A tandem enzyme variants in comparison to the internalization of the non-cytotoxic and RI-sensitive RNase A. After incubation of K-562 cells with the RNase A variants for 36 h, the internalized amount of RNases was analyzed by rapid cell disruption followed by subcellular fractionation and semiquantitative immunoblotting. The data indicate that an enhanced cellular uptake and an increased entry of the RNases into the cytosol can outweigh the abolishment of catalytic activity by RI. As all RNase A variants proved to be resistant to the proteases present in the different subcellular fractions for more than 100 h, our results suggest that the cytotoxic potency of RNases is determined by an efficient internalization into the cytosol.

Role of catalytic and non-catalytic subsite residues in ribonuclease activity of human eosinophil-derived neurotoxin

2009 ◽  
Vol 390 (3) ◽  
Author(s):  
Deepa Sikriwal ◽  
Divya Seth ◽  
Janendra K. Batra
Keyword(s):  
Catalytic Activity ◽  
Biological Activities ◽  
Secretory Protein ◽  
Rnase A ◽  
Phosphate Binding ◽  
Human Eosinophil ◽  
Ribonucleolytic Activity ◽  
Phosphoryl Groups ◽  
Binding Subsites

Abstract Human eosinophil-derived neurotoxin (EDN), a secretory protein from eosinophils, is a member of the RNase A superfamily. The ribonucleolytic activity of EDN is central to its biological activities. EDN binds RNA in a cationic cleft, and the interaction between EDN and RNA substrate extends beyond the scissile bond. Based on its homology with RNase A, putative substrate binding subsites have been identified in EDN. The B1 and B2 subsites interact specifically with bases, whereas P0, P1, and P2 subsites interact with phosphoryl groups. In this study, we evaluated the role of putative residues of these subsites in the ribonucleolytic activity of EDN. We demonstrate that of the two base binding subsites, B1 is critical for the catalytic activity of EDN, as the substrate cleavage was dramatically reduced upon substitution of B1 subsite residues. Among the phosphate-binding subsites, P1 is the most crucial as mutations of its constituting residues totally abolished the catalytic activity of EDN. Mutation of P0 and P2 subsite residues only affected the catalytic activity on the homopolymer Poly(U). Our study demonstrates that P1 and B1 subsites of EDN are critical for its catalytic activity and that the other phosphate-binding subsites are involved in the activity on long homopolymeric substrates.

Total Chemical Synthesis of the Enzyme Sortase AΔN59with Full Catalytic Activity

2014 ◽  
Vol 53 (18) ◽  
pp. 4662-4666 ◽  
Author(s):  
Fang-Kun Deng ◽  
Liang Zhang ◽  
Ya-Ting Wang ◽  
Olaf Schneewind ◽  
Stephen B. H. Kent
Keyword(s):  
Catalytic Activity ◽  
Chemical Synthesis

Expression of adenylate kinase fused mouse ubiquitin active enzyme in Escherichia coli and its application in ubiquitination

2021 ◽  
Author(s):  
Xiaoliang Liu ◽  
Ling Hu ◽  
Yuan Zhang ◽  
Hongtao Li
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Chemical Synthesis ◽  
Adenylate Kinase ◽  
Energy Supply ◽  
Expression System ◽  
Synthesis Process ◽  
Cellular Functions ◽  
Escherichia Coli Expression

Abstract Ubiquitination, is involved in the regulation of numerous cellular functions. Researches in the ubiquitin realm rely heavily on ubiquitination assays in vitro and require large amounts of ubiquitin-activating enzyme (UBA1) and keep ATP supplies. But UBA1 is hard to be obtained with large quantities using reported methods. We fused Escherichia coli adenylate kinase (adk) and mouse UBA1 obtained fusion protein adk-mUBA1. The expression level of adk-mUBA1 increased about 8-fold than that of mUBA1 in Escherichia coli expression system, and adk-mUBA1 was easily purified to 90% purity via two purification steps. The purified adk-mUBA1 protein was functional for ubiquitination and could use ATP in addition to ADP as energy supply and had a higher catalytic activity than mUBA1 in cell lysis. Adk-mUBA1 can be applied to preparing ubiquitin modified substrates and kinds of ubiquitin chains in chemical synthesis process and is preferable application than mUBA1 in vitro ubiquitination.

Role of the N Terminus in RNase A Homologues: Differences in Catalytic Activity, Ribonuclease Inhibitor Interaction and Cytotoxicity

1996 ◽  
Vol 257 (5) ◽  
pp. 992-1007 ◽  
Author(s):  
Ester Boix ◽  
YouNeng Wu ◽  
Veena M. Vasandani ◽  
Shailendra K. Saxena ◽  
Wojciech Ardelt ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Rnase A ◽  
Ribonuclease Inhibitor ◽  
N Terminus ◽  
Inhibitor Interaction

scholarly journals Probing the active site of glyoxalase I from human erythrocytes by use of the strong reversible inhibitor S-p-bromobenzylglutathione and metal substitutions

1981 ◽  
Vol 197 (1) ◽  
pp. 67-75 ◽  
Author(s):  
Anne-Charlotte Aronsson ◽  
Siv Sellin ◽  
Gudrun Tibbelin ◽  
Bengt Mannervik
Keyword(s):  
Catalytic Activity ◽  
Linear Regression Analysis ◽  
Strong Support ◽  
Human Erythrocytes ◽  
Reversible Inhibitor ◽  
Tryptophan Residue ◽  
Glyoxalase I ◽  
Enzyme Molecule ◽  
Binding Parameters ◽  
Thiol Groups

Glyoxalase I from human erythrocytes was studied by use of the strong reversible competitive inhibitor S-p-bromobenzylglutathione. Replacements of cobalt, manganese and magnesium for the essential zinc in the enzyme were made by a new procedure involving 10% methanol as a stabilizer of the enzyme. The Km value for the adduct of methylglyoxal and glutathione was essentially unchanged by the metal substitutions, whereas the inhibition constant for S-p-bromobenzylglutathione increased from 0.08μm for the Zn-containing enzyme to 1.3, 1.7 and 2.4μm for Co-, Mn- and Mg-glyoxalase I respectively. Binding of the inhibitor to the enzyme caused quenching of the tryptophan fluorescence of the protein, from which the binding parameters could be determined by the use of non-linear regression analysis. The highest dissociation constant was obtained for apoenzyme (6.9μm). The identity of the corresponding kinetic and binding parameters of the native enzyme and the Zn2+-re-activated apoenzyme and the clear differences from the parameters of the other metal-substituted enzyme forms give strong support to the previous identification of zinc as the natural metal cofactor of glyoxalase I. Binding to apoenzyme was also shown by the use of S-p-bromobenzylglutathione as a ligand in affinity chromatography and as a protector in chemical modification experiments. The tryptophan-modifying reagent 2-hydroxy-5-nitrobenzyl bromide caused up to 85% inactivation of the enzyme. After blocking of the thiol groups (about 8 per enzyme molecule) 6.1 2-hydroxy-5-nitrobenzyl groups were incorporated. Inclusion of S-p-bromobenzylglutathione with the modifying reagent preserved the catalytic activity of the enzyme completely and decreased the number of modified residues to 4.4 per enzyme molecule. The findings indicate the presence of one tryptophan residue in the active centre of each of the two subunits of the enzyme. Thiol groups appear not to be essential for catalytic activity. The presence of at least two categories of tryptophan residues in the protein was also shown by quenching of the fluorescence by KI.

scholarly journals Copper-mediated selenazolidine deprotection enables one-pot chemical synthesis of challenging proteins

2019 ◽  
Author(s):  
Zhenguang Zhao ◽  
Norman Metanis
Keyword(s):  
Protein Synthesis ◽  
Catalytic Activity ◽  
Chemical Synthesis ◽  
Total Synthesis ◽  
Active Site ◽  
Synthetic Approach ◽  
One Pot ◽  
Chemical Protein Synthesis ◽  
Key Technologies ◽  
The One

<p>While chemical protein synthesis (CPS) has granted access to challenging proteins, synthesis of longer proteins is often limited by low abundance or non-strategic placement of cysteine (Cys) residues, essential for native chemical ligations (NCL), as well as multiple purification and isolation steps. Selective deselenization and one-pot CPS serve as key technologies to circumvent these issues. Herein, we describe the one-pot total synthesis of human thiosulfate: glutathione sulfurtransferase (TSTD1), a 115-residue protein with a single Cys residue at its active site, and its seleno-analogue. WT-TSTD1 was synthesized in a C-to-N synthetic approach employing multiple NCL reactions, Cu(II)-mediated deprotection of selenazolidine (Sez), and chemoselective deselenization, all in one-pot. In addition, the protein’s seleno analogue (Se-TSTD1), in which the active site Cys is replaced with selenocysteine, was synthesized with a kinetically controlled ligation in a one-pot, N-to-C synthetic approach. TSTD1’s one-pot synthesis was made possible by the newly reported, rapid, and facile copper-mediated selenazolidine deprotection that can be accomplished in one minute. Finally, catalytic activity of the two proteins indicated that Se-TSTD1 possessed only four-fold lower activity than WT-TSTD1 as a thiosulfate: glutathione sulfurtransferase, suggesting that selenoproteins can have physiologically comparable sulfutransferase activity as their cysteine counterparts. </p>

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