Characterization of bromosulphophthalein binding to human glutathione S-transferase A1-1: thermodynamics and inhibition kinetics

In addition to their catalytic functions, GSTs (glutathione S-transferases) bind a wide variety of structurally diverse non-substrate ligands. This ligandin function is known to result in the inhibition of catalytic function. The interaction between hGSTA1-1 (human class Alpha GST with two type 1 subunits) and a non-substrate anionic ligand, BSP (bromosulphophthalein), was studied by isothermal titration calorimetry and inhibition kinetics. The binding isotherm is biphasic, best described by a set of two independent sites: a high-affinity site and a low-affinity site(s). The binding stoichiometries for these sites are 1 and 3 molecules of BSP respectively. BSP binds to the high-affinity site 80 times more tightly (Kd=0.12 μM) than it does to the low-affinity site(s) (Kd=9.1 μM). Binding at these sites is enthalpically and entropically favourable, with no linkage to protonation events. Temperature- and salt-dependent studies indicate the significance of hydrophobic interactions in the binding of BSP, and that the low-affinity site(s) displays low specificity towards the anion. Binding of BSP results in the release of ordered water molecules at these hydrophobic sites, which more than offsets unfavourable entropic changes during binding. BSP inhibition studies show that the binding of BSP to its high-affinity site does not inhibit hGSTA1-1. This site, located near Trp-20, may be related to the buffer-binding site observed in GSTP1-1. The low-affinity-binding site(s) for BSP is most probably located at or near the active site of hGSTA1-1. Binding to this site(s) results in non-competitive inhibition with respect to CDNB (1-chloro-2,4-dinitrobenzene) (KiBSP=16.8±1.9 μM). Given the properties of the H site and the relatively small size of the electrophilic substrate CDNB, it is plausible that the active site of the enzyme can simultaneously accommodate both BSP and CDNB. This would explain the non-competitive behaviour of certain inhibitors that bind the active site (e.g. BSP).

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Binding of [14C]malonyl-CoA to rat liver mitochondria after blocking of the active site of carnitine palmitoyltransferase I. Displacement of low-affinity binding by palmitoyl-CoA

Biochemical Journal ◽

10.1042/bj2330589 ◽

1986 ◽

Vol 233 (2) ◽

pp. 589-593 ◽

Cited By ~ 17

Author(s):

B D Grantham ◽

V A Zammit

Keyword(s):

Binding Site ◽

Rat Liver ◽

Active Site ◽

Liver Mitochondria ◽

Carnitine Palmitoyltransferase ◽

Rat Liver Mitochondria ◽

High Affinity ◽

Malonyl Coa ◽

High Affinity Site ◽

Cpt I

The active site of the overt activity of carnitine palmitoyltransferase (CPT I) in rat liver mitochondria was blocked by the self-catalysed formation of the S-carboxypalmitoyl-CoA ester of (-)-carnitine, followed by washing of the mitochondria. CPT I activity in treated mitochondria was inhibited by 90-95%. Binding of [14C]malonyl-CoA to these mitochondria was not inhibited as compared with that of control mitochondria. When CPT I activity was inhibited, palmitoyl-CoA could markedly displace [14C]malonyl-CoA binding from the low-affinity site for the inhibitor [Zammit, Corstorphine & Gray (1984) Biochem. J. 222, 335-342], but not from the high-affinity site for malonyl-CoA binding. The saturation characteristics of the malonyl-CoA-binding component lost in the presence of palmitoyl-CoA were sigmoidal, and thus suggestive of co-operative binding at this site. It is suggested that the site hitherto considered to be a low-affinity malonyl-CoA-binding site may be effectively a second, allosteric, acyl-CoA-binding site on CPT I under conditions that prevail in vivo, whereas the high-affinity site for malonyl-CoA may be exclusive to the inhibitor. The possibility that the competitive-type interactions of malonyl-CoA and acyl-CoA on CPT I activity could arise from the effects of separate malonyl-CoA and acyl-CoA allosteric sites is considered. The possible significance of the large difference in the capacity of the two sites and their different saturation kinetics is also discussed.

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Design and Characterisation of Inhibitory Peptides against Bleg1_2478, an Evolutionary Divergent B3 Metallo-β-lactamase

Molecules ◽

10.3390/molecules25245797 ◽

2020 ◽

Vol 25 (24) ◽

pp. 5797

Author(s):

Gayathri Selvaraju ◽

Thean Chor Leow ◽

Abu Bakar Salleh ◽

Yahaya M. Normi

Keyword(s):

Active Site ◽

Protein Complexes ◽

Hypothetical Protein ◽

Titration Calorimetry ◽

Binding Properties ◽

Vitro Assay ◽

In Silico Docking ◽

Inhibitory Peptides ◽

Catalytic Function

Previously, a hypothetical protein (HP) termed Bleg1_2437 (currently named Bleg1_2478) from Bacillus lehensis G1 was discovered to be an evolutionary divergent B3 subclass metallo-β-lactamase (MBL). Due to the scarcity of clinical inhibitors for B3 MBLs and the divergent nature of Bleg1_2478, this study aimed to design and characterise peptides as inhibitors against Bleg1_2478. Through in silico docking, RSWPWH and SSWWDR peptides with comparable binding energy to ampicillin were obtained. In vitro assay results showed RSWPWH and SSWWDR inhibited the activity of Bleg1_2478 by 50% at concentrations as low as 0.90 µM and 0.50 µM, respectively. At 10 µM of RSWPWH and 20 µM of SSWWDR, the activity of Bleg1_2478 was almost completely inhibited. Isothermal titration calorimetry (ITC) analyses showed slightly improved binding properties of the peptides compared to ampicillin. Docked peptide–protein complexes revealed that RSWPWH bound near the vicinity of the Bleg1_2478 active site while SSWWDR bound at the center of the active site itself. We postulate that the peptides caused the inhibition of Bleg1_2478 by reducing or blocking the accessibility of its active site from ampicillin, thus hampering its catalytic function.

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A gonadotropin-releasing hormone type neuropeptide with a high affinity binding site for copper(ii) and nickel(ii)

Metallomics ◽

10.1039/c8mt00279g ◽

2019 ◽

Vol 11 (2) ◽

pp. 404-414 ◽

Cited By ~ 5

Author(s):

Kevin K. Tran ◽

Bhawantha M. Jayawardena ◽

Maurice R. Elphick ◽

Christopher E. Jones

Keyword(s):

Binding Site ◽

Gonadotropin Releasing Hormone ◽

Affinity Binding ◽

Asterias Rubens ◽

High Affinity Binding ◽

Releasing Hormone ◽

High Affinity ◽

Evolutionarily Conserved ◽

High Affinity Site ◽

High Affinity Binding Site

Gonadotropin releasing hormone from Asterias rubens binds Cu(ii) in a nitrogen-rich, high-affinity site. Cu(ii)-binding is an evolutionarily conserved feature of GnRH-type neuropeptides.

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The binding of [3H]adenosine to synaptosomal and other preparations from the mammalian brain

Biochemical Journal ◽

10.1042/bj1940611 ◽

1981 ◽

Vol 194 (2) ◽

pp. 611-620 ◽

Cited By ~ 36

Author(s):

M E Newman ◽

J Patel ◽

H McIlwain

Keyword(s):

Binding Site ◽

Adenosine Deaminase ◽

Binding Capacity ◽

Receptor Site ◽

Mammalian Brain ◽

Affinity Binding ◽

Temperature Dependent ◽

High Affinity ◽

High Affinity Site ◽

Uptake Process

1. A high-affinity adenosine-binding site with Kd(adenosine) 0.5-1.3 microM was demonstrated in particulate and synaptosomal fractions isolated from the cerebral cortex of guinea pig, rat and ox. 2. Binding of [3H]adenosine to this site was inhibited by theophylline and by 2-chloroadenosine, but not by four other adenosine analogues. 3. Endogenous adenosine, found to be present in some preparations at approx. 1 pmol/mg of protein, diminished the binding capacity of the preparations for [3H]adenosine. 4. Addition of the adenosine deaminase inhibitor erythro-9-[1-(1-hydroxyethyl)heptyl]-adenine revealed the presence of a second lower affinity binding site with Kd (adenosine) 5-9 microM and a higher maximal adenosine-binding capacity. The inhibitor partially blocked binding to the high-affinity site in preparations from which adenosine deaminase had been removed by washing. 5. To preparations of particulate fractions maintained under iso-osmotic conditions, adenosine attachment was non-saturable and temperature-dependent, indicating the existence of an active uptake process. 6. The location and binding constant of the high-affinity adenosine-binding site suggest that it corresponds to the receptor site for adenosine-activated adenylate cyclase.

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Powerful Induction of Divergent tgs1-Rv3131 Genes in Mycobacterium tuberculosis Is Mediated by DevR Interaction with a High-Affinity Site and an Adjacent Cryptic Low-Affinity Site

Journal of Bacteriology ◽

10.1128/jb.00310-09 ◽

2009 ◽

Vol 191 (19) ◽

pp. 6075-6081 ◽

Cited By ~ 24

Author(s):

Santosh Chauhan ◽

Jaya Sivaswami Tyagi

Keyword(s):

Mycobacterium Tuberculosis ◽

Binding Site ◽

Binding Sites ◽

Target Genes ◽

Promoter Activation ◽

Transcription Start ◽

High Affinity ◽

Synergistic Activation ◽

Dnase I Footprinting ◽

High Affinity Site

ABSTRACT DevR activates the transcription of ∼48 genes in response to hypoxia and other stresses and triggers metabolic downshift and dormancy development in Mycobacterium tuberculosis. tgs1 and Rv3131 encode triacylglycerol synthase and a putative nitroreductase, respectively, and both are members of the DevR regulon. This study aimed to understand how a single putative DevR binding site identified previously could sustain powerful induction of divergent tgs1-Rv3131 genes. DNase I footprinting revealed that phosphorylated DevR in fact binds to two sites symmetrically located at −42.5 and −63.5 bp from transcription start points of both genes. DevR first bound to the high-affinity site, P, and cooperatively recruited another DevR molecule to the secondary low-affinity site, S, to activate tgs1-Rv3131 transcription by ∼210- and ∼110-fold, respectively. The presence of a single P site significantly reduced activation of tgs1 expression and abolished Rv3131 activity, reinforcing the requirement of two binding sites for robust expression in both directions. P site inversion abolished tgs1 but not Rv3131 transcription despite DevR occupancy at both sites. The lack of tgs1 expression is most likely due to disruption of its −35 promoter element rather than inversion of the binding site per se. We conclude that (i) an overlap of a DevR binding site and −35 sequence is indispensable for promoter activation, (ii) DevR interaction with two binding sites is obligatory for synergistic activation of tgs1-Rv3131 promoters, and (iii) DevR interaction with binding sites of different affinities offers scope for temporal and differential expression of target genes.

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Enthalpic cost of water removal from a glucose binding cavity on HK620 TSP

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314083995 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1600-C1600

Author(s):

Ulrich Gohlke ◽

Nina Broeker ◽

Udo Heinemann ◽

Robert Seckler ◽

Stefanie Barbirz

Keyword(s):

Complex Formation ◽

Binding Site ◽

Dissociation Constants ◽

Point Mutations ◽

Crystal Structure Analysis ◽

Titration Calorimetry ◽

High Affinity ◽

Sugar Binding ◽

Glucose Binding ◽

Binding Cavity

Bacteriophage HK620 recognizes and cleaves the O-antigen polysaccharide of Escherichia coli serogroup O18A1 with its tailspike protein (TSP). HK620TSP binds hexasaccharide fragments with low affinity, but single and double amino acid exchanges generated a set of high-affinity mutants with submicromolar dissociation constants. Isothermal titration calorimetry showed that only small amounts of heat were released upon complex formation via a large number of direct and solvent-mediated hydrogen bonds between carbohydrate and protein. At room temperature, association was both enthalpy- and entropy-driven, emphasizing major solvent rearrangements upon complex formation. Crystal structure analysis of a complete set of combinations of wildtype protein and point mutations with and without polysaccharide ligands was carried out. It could be shown that the extended sugar binding site can be dissected into two regions: first, a hydrophobic pocket at the reducing end with minor affinity contributions. Second, a region where the specific exchange of amino acids creates a site for additional water molecules. Sidechain rearrangements upon sugar binding lead to desolvation and additional hydrogen bonding which define this region of the binding site as the high-affinity scaffold.

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Alteration of the Factor X Zymogen to Protease Transition Provides Evidence for Allosteric Linkage between the S1 and FVa Binding Sites.

Blood ◽

10.1182/blood.v106.11.30.30 ◽

2005 ◽

Vol 106 (11) ◽

pp. 30-30 ◽

Cited By ~ 1

Author(s):

Raffaella Toso ◽

Hua Zhu ◽

Rodney M. Camire

Keyword(s):

Binding Site ◽

Active Site ◽

Membrane Surface ◽

Coagulation Factor ◽

Salt Bridge ◽

Kinetic Studies ◽

Factor X ◽

Bridge Formation ◽

High Affinity ◽

Km Value

Abstract The zymogen to protease transition in the chymotrypsin-like serine protease family follows a well described mechanism in which bond cleavage at a highly conserved site (Arg15-Ile16; chymotrypsin numbering system) results in the unmasking of a new N-terminus that acts as an intramolecular ligand for Asp194. This new salt-bridge drives a conformational change in the so-called “activation domain”, surface loops consisting of the S1 specificity pocket, oxyanion hole, autolysis loop, and sodium biding site. It is well documented in the trypsin system that Ile16-Asp194 internal salt-bridge formation is allosterically linked to the S1 specificity site; that is changes at one site influence the other and vice versa. Blood coagulation factor Xa (FXa) reversibly associates with its cofactor factor Va (FVa) on a membrane surface in the presence of Ca2+ ions with high affinity; an interaction which is not mimicked by the zymogen FX. To determine whether the FX zymogen to protease transition contributes to the expression of a high affinity FVa binding site, we constructed a series of FXa variants which are shifted along this transition pathway. To generate these “zymogen-like” proteins, we made several substitutions at position 16 or 17, with the intent of destabilizing the intramolecular salt bridge to varying degrees. Following a series of preliminary experiments, three mutants were chosen for expression, purification, and activation with RVV-X: I16L, I16G, and V17A. Kinetic studies using peptidyl substrates and active site directed probes revealed that I16L and V17A have an impaired ability to bind these probes (15 to 25-fold increase in the Km or Ki) while the rate of catalysis (kcat) was reduced by 3-fold compared to wild-type FXa (wtFXa; plasma-derived and recombinant). The I16G variant was not inhibited by any of the probes examined and its chromogenic activity was severely impaired (>500 to 1000-fold), precluding calculation of kinetic parameters. These data are consistent with the idea that destabilization of internal salt-bridge formation (Ile16-Asp194) influences binding at the S1 specificity site. In contrast to these results, assembly of I16L and V17A into prothrombinase almost completely restored the Km for peptidyl substrates while the kcat was still 3-fold reduced, indicating that FVa binding can rescue binding at the active site. Surprisingly, even the Km value for I16G was almost completely restored (3-fold increased compared to wtFXa) when assembled in prothrombinase; however a 60-fold reduction in the kcat was found. Consistent with these data, kinetic studies using prothrombin or prethrombin-1 revealed that each of the FXa variants had a normal Km value when assembled in prothrombinase; while the kcat values where reduced to a similar extent as for the chromogenic substrates. Overall our data indicate that direct binding of these FXa variants to FVa rescues binding at S1 site, suggesting allosteric linkage exists between these sites. Thus the FX zymogen to protease transition not only influences the formation of the S1 pocket, but also contributes in a substantial way to the formation of a FVa binding site.

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Kinetic and structural characterization of a product complex of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Escherichia coli

Biochemical Journal ◽

10.1042/bj20031850 ◽

2004 ◽

Vol 380 (3) ◽

pp. 867-873 ◽

Cited By ~ 9

Author(s):

Arnaud GARÇON ◽

Alun BERMINGHAM ◽

Lu-Yun LIAN ◽

Jeremy P. DERRICK

Keyword(s):

Binding Site ◽

Quantum Coherence ◽

Chemical Shifts ◽

Titration Calorimetry ◽

Second Phase ◽

Transient Kinetics ◽

Antimicrobial Drugs ◽

High Affinity ◽

High Affinity Binding Site ◽

Equilibrium Dissociation

HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase) catalyses the transfer of pyrophosphate from ATP to HMDP (6-hydroxymethyl-7,8-dihydropterin), to form AMP and DHPPP (6-hydroxymethyl-7,8-dihydropterin pyrophosphate). This transformation is a key step in the biosynthesis of folic acid, and HPPK is consequently a target for antimicrobial drugs. The substrates are known to bind to HPPK in an ordered manner, with ATP binding first followed by HMDP. In the present study we show by isothermal titration calorimetry that the product, DHPPP, can bind to the HPPK apoenzyme with high affinity (equilibrium dissociation constant, Kd=0.2 µM), but without the enhancement of pterin fluorescence that occurs on binding of HMDP. The transient kinetics of the enzyme can be monitored by measuring the change in the fluorescence of the pterin ring using stopped-flow methods. The fluorescence exhibits a pronounced biphasic behaviour: it initially rises and then declines back to its original level. This behaviour is in agreement with a two-state kinetic model, with the first phase of fluorescence increase associated with HMDP binding to the enzyme, and the second phase with a slow event that occurs after the reaction has taken place. The HPPK–DHPPP and HPPK–DHPPP–AMP complexes were examined by NMR, and the binding site for DHPPP partially mapped from changes in chemical shifts identified from two dimensional 1H/15N heteronuclear single-quantum coherence spectra. The results demonstrate that DHPPP, in contrast to HMDP, is able to bind to the HPPK apoenzyme and suggest that the pyrophosphate moieties on the ligand play an important role in establishment of a high affinity binding site for the pterin ring.

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Interaction of Echicetin with a High Affinity Thrombin Binding Site on Platelet Glycoprotein GPIb

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649854 ◽

1995 ◽

Vol 74 (03) ◽

pp. 954-957 ◽

Cited By ~ 20

Author(s):

Manling Peng ◽

Frances A Emig ◽

Ahua Mao ◽

Weiqi Lu ◽

Edward P Kirby ◽

...

Keyword(s):

Platelet Aggregation ◽

Binding Site ◽

Snake Venom ◽

Platelet Glycoprotein ◽

High Affinity ◽

Low Concentrations ◽

High Affinity Site ◽

Echis Carinatus

SummaryEchicetin, a protein isolated from Echis carinatus snake venom, inhibited platelet aggregation and secretion induced by low concentrations of thrombin (<0.2 U/ml), by binding to platelet glycoprotein lb (GPIb). The inhibition was not observed when the platelets were stimulated with higher concentrations of thrombin (>0.2 U/ml). Echicetin competed with thrombin for binding to the high affinity site on GPIb. Thrombin also inhibited 50% of the binding of 125I-echicetin to the platelets.

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Studies on the mechanism of action of a bilayer stabilizing inhibitor of protein kinase C: Cholesterylphosphoryldimethylethanolamine

Bioscience Reports ◽

10.1007/bf01114684 ◽

1989 ◽

Vol 9 (3) ◽

pp. 315-328 ◽

Cited By ~ 7

Author(s):

Richard M. Epand ◽

Alan R. Stafford ◽

Remo Bottega ◽

Eric H. Ball

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Active Site ◽

Potent Inhibitor ◽

Competitive Inhibition ◽

Inhibition Kinetics ◽

Kinase C ◽

Triton X ◽

Kinetics Of ◽

Tryptic Hydrolysis

Cholesterylphosphoryldimethylethanolamine is a zwitterionic compound which is a good bilayer stabilizer. As has been found with many other compounds having these properties, cholesterylphosphoryldimethylethanolamine is found to be a potent inhibitor of protein kinase C in both vesicle and micelle assay systems. The kinetics of the inhibition in Triton X-100 micelles was non-competitive with respect to ATP, histone, diolein, phorbol ester and Ca2+. It has a Ki of about 30 μm. The inhibition kinetics as a function of phosphatidylserine concentration is more complex but suggestive of competitive inhibition. Cholesterylphosphoryldimethylethanolamine does not prevent the partitioning of protein kinase C into the membrane. This inhibitor lowers the Ca2+-phosphatidylserine-independent phosphorylation of protamine sulfate by protein kinase C and directly affects the catalytic segment of the enzyme generated by tryptic hydrolysis. Thus, this zwitterionic bilayer stabilizing inhibitor of protein kinase C both competes with the binding of phosphatidylserine as well as affects the active site of protein kinase C.

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