Mapping the active site of papain with the aid of peptide substrates and inhibitors

1970 ◽  
Vol 257 (813) ◽  
pp. 249-264 ◽  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Amino Acid Residue ◽  
Active Site ◽  
Proteolytic Enzymes ◽  
Reaction Rates ◽  
Side Chain ◽  
Peptide Substrates ◽  
Methyl Group ◽  
Kinetics Of

The active site of an enzyme performs the twofold function of binding a substrate and catalysing a reaction. The efficiency of these actions determines the overall activity of the enzyme towards the particular substrate, i.e. determines the specificity of the enzyme. It is therefore possible to obtain information on the active site by the kinetics of the enzyme’s reactions with different substrates and inhibitors. An important feature of the active site is its size. It should be possible to 'measure’ this by using substrates or inhibitors large enough to show up the interactions of the furthermost parts of the binding site. In the present series of investigations on proteolytic enzymes, our approach is to compare the activity of the enzyme towards ( a ) peptides of increasing length, ( b ) diastereoisomeric pairs of peptides in which a particular amino acid residue has been replaced by its antipode, and ( c ) pairs of substrates in which a particular side chain (say a methyl group) has been replaced by another (say an aromatic group). The influence of these changes on reaction rates as a function of distance from the point of cleavage indicates the extent of the active site (Schechter, Abramowitz & Berger 1965; Abramowitz, Schechter & Berger 1967).

scholarly journals Affinity-labelling of the anti-inflammatory drug and prostaglandin-binding site of 3 α-hydroxysteroid dehydrogenase of rat liver cytosol with 17 β- and 21-bromoacetoxysteroids

1987 ◽  
Vol 245 (1) ◽  
pp. 269-276 ◽  
Author(s):  
T M Penning ◽  
K E Carlson ◽  
R B Sharp
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Rat Liver ◽  
Active Site ◽  
Hydroxysteroid Dehydrogenase ◽  
Side Chain ◽  
Liver Cytosol ◽  
Affinity Labelling ◽  
Anti Inflammatory ◽  
Rat Liver Cytosol

The homogeneous 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol binds prostaglandins with low micromolar affinity at its active site and is competitively inhibited by the non-steroidal and steroidal anti-inflammatory drugs [Penning, Mukharji, Barrows & Talalay (1984) Biochem. J. 222, 601-611]. To examine the portion of this binding site that accommodates the glucocorticoid side chain, we have synthesized 17 beta-bromoacetoxy-5 alpha-dihydrotestosterone (BrDHT) and 21-bromoacetoxydesoxycorticosterone (BrDOC) as affinity-labelling agents. Both these agents promote rapid inactivation of the purified enzyme in a time- and concentration-dependent manner. Analyses of the inactivation progress curves gave estimates of Ki for the inactivators and half-life (t1/2) for the enzyme at saturation (tau) as follows: Ki = 33 microM and tau = 18 s for BrDHT, and Ki = 10 microM and tau = 203 s for BrDOC. Under initial-velocity conditions BrDHT and BrDOC act as competitive inhibitors, yielding Ki values identical with those measured in the inactivation experiments. Both indomethacin and prostaglandin E2 can protect the enzyme from inactivation, yielding Ki values for these ligands consistent with those measured independently by competitive-inhibition studies. These data confirm that the bromoacetoxysteroids label the active site, which is coincident with the prostaglandin- and anti-inflammatory-drug-binding site. Neither gel filtration nor extensive dialysis restores activity to the enzyme inactivated with either affinity-labelling agent. Use of radioactive BrDHT or BrDOC, in which either the steroid portion is labelled with 3H or the bromoacetate portion is labelled with 14C, indicates that inactivation is accompanied by a stoichiometric incorporation of 0.7-1.0 molecules of inhibitor per enzyme monomer. The linkage that forms between the dehydrogenase with either [14C]BrDHT or [14C]BrDOC is stable to acid and base treatment. Complete acid hydrolysis of the enzyme inactivated with [14C]BrDHT, followed by amino acid analyses, indicates that 87% of the radioactivity is eluted with carboxymethylcysteine. An almost identical result is obtained with [14C]BrDOC, where at least 75% of the radioactivity is eluted with this amino acid. Thus BrDHT and BrDOC alkylate at least one reactive cysteine residue at the active site that may be of functional importance in binding the glucocorticoid side chain.

Schistosomal Sulfotransferase Interaction with Oxamniquine Involves Hybrid Mechanism of Induced-fit and Conformational Selection

2020 ◽  
Vol 16 (4) ◽  
pp. 451-459 ◽  
Author(s):  
Fortunatus C. Ezebuo ◽  
Ikemefuna C. Uzochukwu
Keyword(s):  
Molecular Dynamics ◽  
Amino Acid ◽  
Binding Energy ◽  
Binding Site ◽  
Conformational Dynamics ◽  
Side Chain ◽  
Amino Acid Residues ◽  
Conformational Selection ◽  
Hybrid Mechanism ◽  
Dynamics Simulations

Background: Sulfotransferase family comprises key enzymes involved in drug metabolism. Oxamniquine is a pro-drug converted into its active form by schistosomal sulfotransferase. The conformational dynamics of side-chain amino acid residues at the binding site of schistosomal sulfotransferase towards activation of oxamniquine has not received attention. Objective: The study investigated the conformational dynamics of binding site residues in free and oxamniquine bound schistosomal sulfotransferase systems and their contribution to the mechanism of oxamniquine activation by schistosomal sulfotransferase using molecular dynamics simulations and binding energy calculations. Methods: Schistosomal sulfotransferase was obtained from Protein Data Bank and both the free and oxamniquine bound forms were subjected to molecular dynamics simulations using GROMACS-4.5.5 after modeling it’s missing amino acid residues with SWISS-MODEL. Amino acid residues at its binding site for oxamniquine was determined and used for Principal Component Analysis and calculations of side-chain dihedrals. In addition, binding energy of the oxamniquine bound system was calculated using g_MMPBSA. Results: The results showed that binding site amino acid residues in free and oxamniquine bound sulfotransferase sampled different conformational space involving several rotameric states. Importantly, Phe45, Ile145 and Leu241 generated newly induced conformations, whereas Phe41 exhibited shift in equilibrium of its conformational distribution. In addition, the result showed binding energy of -130.091 ± 8.800 KJ/mol and Phe45 contributed -9.8576 KJ/mol. Conclusion: The results showed that schistosomal sulfotransferase binds oxamniquine by relying on hybrid mechanism of induced fit and conformational selection models. The findings offer new insight into sulfotransferase engineering and design of new drugs that target sulfotransferase.

scholarly journals Effect of roasting temperature on lipid and protein oxidation and amino acid residue side chain modification of beef patties

RSC Advances ◽  
2021 ◽  
Vol 11 (35) ◽  
pp. 21629-21641
Author(s):  
Chao Xia ◽  
Pingping Wen ◽  
Yaming Yuan ◽  
Xiaofan Yu ◽  
Yijing Chen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Protein Oxidation ◽  
Relative Number ◽  
Side Chain ◽  
Amino Acid Residues ◽  
Beef Patties ◽  
Lipid And Protein Oxidation ◽  
Roasting Temperature

The relative number of peptides modified by the amino acid residues of actin from raw beef patties and those cooked at different roasting temperatures.

scholarly journals Irreversible site-directed labeling of the 4-aminobutyrate binding site by tritiated meta-sulfonate benzene diazonium . Contribution of a nucleophilic amino acid residue of the alpha1 subunit

1999 ◽  
Vol 265 (1) ◽  
pp. 189-194 ◽  
Author(s):  
Patrice Jacques ◽  
Philippe Perret ◽  
Marie-Jeanne Bouchet ◽  
Bernard Foucaud ◽  
Maurice Goeldner ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Amino Acid Residue ◽  
Benzene Diazonium

scholarly journals Monitoring the stabilities of a mixture of peptides by mass-spectrometry-based techniques

2019 ◽  
Vol 25 (1) ◽  
pp. 73-81 ◽  
Author(s):  
Daniel R Fuller ◽  
Christopher R Conant ◽  
Tarick J El-Baba ◽  
Zhichao Zhang ◽  
Kameron R Molloy ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Transition State ◽  
Elevated Temperatures ◽  
Proteolytic Enzymes ◽  
Side Chain ◽  
Free Energies ◽  
Dissociation Kinetics ◽  
Penultimate Proline ◽  
The Stability

Biomolecular degradation plays a key role in proteostasis. Typically, proteolytic enzymes degrade proteins into smaller peptides by breaking amino acid bonds between specific residues. Cleavage around proline residues is often missed and requires highly specific enzymes for peptide processing due to the cyclic proline side-chain. However, degradation can occur spontaneously (i.e. in the absence of enzymes). In this study, the influence of the first residue on the stability of a series of penultimate proline containing peptides, with the sequence Xaa–Pro–Gly–Gly (where Xaa is any amino acid), is investigated with mass spectrometry techniques. Peptides were incubated as mixtures at various solution temperatures (70℃ to 90℃) and were periodically sampled over the duration of the experiment. At elevated temperatures, we observe dissociation after the Xaa–Pro motif for all sequences, but at different rates. Transition state thermochemistry was obtained by studying the temperature-dependent kinetics and although all peptides show relatively small differences in the transition state free energies (∼95 kJ/mol), there is significant variability in the transition state entropy and enthalpy. This demonstrates that the side-chain of the first amino acid has a significant influence on the stability of the Xaa–Pro sequence. From these data, we demonstrate the ability to simultaneously measure the dissociation kinetics and relative transition state thermochemistries for a mixture of peptides, which vary only in the identity of the N-terminal amino acid.

Tetranectin-Binding Site on Plasminogen Kringle 4 Involves the Lysine-Binding Pocket and at Least One Additional Amino Acid Residue†

Biochemistry ◽  
2000 ◽  
Vol 39 (25) ◽  
pp. 7414-7419 ◽  
Author(s):  
Jonas H. Graversen ◽  
Bent W. Sigurskjold ◽  
Hans C. Thøgersen ◽  
Michael Etzerodt
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Amino Acid Residue ◽  
Binding Pocket ◽  
Additional Amino Acid

Selective inhibition of dipeptidyl peptidase 4 by targeting a substrate-specific secondary binding site

2011 ◽  
Vol 392 (3) ◽  
Author(s):  
Kerstin Kühn-Wache ◽  
Joachim W. Bär ◽  
Torsten Hoffmann ◽  
Raik Wolf ◽  
Jens-Ulrich Rahfeld ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Specific Activity ◽  
Selective Inhibition ◽  
Dipeptidyl Peptidase ◽  
Peptide Substrates ◽  
Catalytic Center ◽  
Dipeptidyl Peptidase 4 ◽  
Peptide Family ◽  
Specificity Constant

Abstract Dipeptidyl peptidase 4/CD26 (DP4) is a multifunctional serine protease liberating dipeptide from the N-terminus of (oligo)peptides which can modulate the activity of these peptides. The enzyme is involved in physiological processes such as blood glucose homeostasis and immune response. DP4 substrate specificity is characterized in detail using synthetic dipeptide derivatives. The specificity constant k cat/K m strongly depends on the amino acid in P1-position for proline, alanine, glycine and serine with 5.0×105 m -1s-1, 1.8×104 m -1s-1, 3.6×102 m -1s-1, 1.1×102 m -1s-1, respectively. By contrast, kinetic investigation of larger peptide substrates yields a different pattern. The specific activity of DP4 for neuropeptide Y (NPY) cleavage comprising a proline in P1-position is the same range as the k cat/K m values of NPY derivatives containing alanine or serine in P1-position with 4×105 m -1s-1, 9.5×105 m -1s-1 and 2.1×105 m -1s-1, respectively. The proposed existence of an additional binding region outside the catalytic center is supported by measurements of peptide substrates with extended chain length. This ‘secondary’ binding site interaction depends on the amino acid sequence in P4′–P8′-position. Interactions with this binding site could be specifically blocked for substrates of the GRF/glucagon peptide family. By contrast, substrates not belonging to this peptide family and dipeptide derivative substrates that only bind to the catalytic center of DP4 were not inhibited. This more selective inhibition approach allows, for the first time, to distinguish between substrate families by substrate-discriminating inhibitors.

scholarly journals Importance of C-Terminal Knot Structure in Carbonic Anhydrase II (Part I): Role of C-Terminal Knot Forming GLN253 on Enzymatic Property of Human Carbinic II

1970 ◽  
Vol 14 ◽  
pp. 1-9
Author(s):  
Mohammad Taufiq Alam
Keyword(s):  
Amino Acid ◽  
Carbonic Anhydrase ◽  
Point Mutation ◽  
Amino Acid Residue ◽  
Active Site ◽  
Terminal Region ◽  
Carbonic Anhydrase Ii ◽  
C Terminus ◽  
Human Carbonic Anhydrase

In both, bovine and human carbonic anhydrase II, a conserved glutamine residue occupies the position in the middle of the knot, which is formed by intercrossing of C-terminal end with N-terminal region. Previous studies have indicated that C-terminus is not the part of an active site, but truncation of 7 amino acid residue in this region can have marked effects on stability of the enzyme (data not published). To gain further insight into the role of specific amino acid residue in C-terminal region, site directed mutagenesis was used to introduce point mutation. Substitution of glutamine with cysteine was chosen because the cysteine residue is less hydrophilic as compared with glutamine and thus, may disrupt the hydrophilic environment in this region. Result indicates that Gln253 located within the C-terminus knot topology plays a significant role in normal function of the enzyme. Thus, C-terminal region might mediate cooperativity between the central active site of the enzyme through proper formation of knot. Key words: Human carbonic anhydrase II; knot topology; point mutation J. bio-sci. 14: 1-9, 2006

Sign in / Sign up

Export Citation Format

Share Document