scholarly journals Distribution of Charged and Hydrophobic Amino Acids on the Surfaces of Two Types of Beta-Fructosidase from Thermotoga Maritima

2020 ◽  
Vol 2 (1) ◽  
pp. 4
Author(s):  
Farkhat Sakibaev ◽  
Marina Holyavka ◽  
Victoria Koroleva ◽  
Valeriy Artyukhov
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Active Center ◽  
Active Site ◽  
Thermotoga Maritima ◽  
Significant Loss ◽  
Hydrophobic Residues ◽  
Charged Amino Acids ◽  
Hydrophobic Amino Acids

Thermotoga maritima beta-fructosidases are enzymes that release beta-D-fructose from sucrose, raffinose, and fructan polymers such as inulin. The surfaces of beta-fructosidases 1UYP and 1W2T from Thermotoga maritima were studied in this work. It was showed that amino acids are not distributed equally on the surfaces of the enzymes. Several clusters of charged and hydrophobic residues were detected at pH 7.0. Such clusters were detected by calculation of the distances between them. It was determined that on surfaces of beta-fructosidases PDB ID: 1UYP and PDB ID: 1W2T, 96% and 95% of charged amino acids and also 50% and 42% of hydrophobic amino acids form clusters, respectively. Six clusters of charged amino acids on the surface of beta-fructosidase 1UYP and five clusters on the surface of beta-fructosidase 1W2T were detected. The composition of such clusters is presented. Both types of beta-fructosidase have three clusters of hydrophobic amino acids on their surface. These facts should be considered when choosing immobilization conditions. It was shown that a charged matrix is more promising for the immobilization of beta-fructosidases 1UYP and 1W2T from Thermotoga maritima due to the possibility of binding without any significant loss of activity due to their overlapping active center. Hydrophobic carriers are less promising due to the probable active site overlap. Such binding may have a loss of enzyme activity as a result.

scholarly journals Early Experiments in the Quest of an Active Center in Chymotrypsin

1962 ◽  
Vol 45 (4) ◽  
pp. 47-56 ◽  
Author(s):  
A. K. Balls
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Active Center ◽  
Active Site ◽  
Hydrolytic Enzymes

It is now quite proper to speak of an "active site" or "center" in (or on) a number of hydrolytic enzymes, chymotrypsin in particular. Such sites have been clearly demonstrated and in some cases they have even been investigated in detail. We are almost persuaded that they are the actual loci of enzyme activity in nature. Whatever we think of them, we do know that they are "some peculiar arrangement of amino acids" (2 a).

scholarly journals A shift in aggregation avoidance strategy marks a long-term direction to protein evolution

2017 ◽  
Author(s):  
S.G. Foy ◽  
B.A. Wilson ◽  
J. Bertram ◽  
M.H.J. Cordes ◽  
J. Masel
Keyword(s):  
Amino Acids ◽  
Common Ancestor ◽  
Gene Families ◽  
Protein Coding ◽  
Hydrophobic Residues ◽  
Extant Species ◽  
Aggregation Problem ◽  
Hydrophobic Amino Acids ◽  
Ancestral States

AbstractTo detect a direction to evolution, without the pitfalls of reconstructing ancestral states, we need to compare “more evolved” to “less evolved” entities. But because all extant species have the same common ancestor, none are chronologically more evolved than any other. However, different gene families were born at different times, allowing us to compare young protein-coding genes to those that are older and hence have been evolving for longer. To be retained during evolution, a protein must not only have a function, but must also avoid toxic dysfunction such as protein aggregation. There is conflict between the two requirements; hydrophobic amino acids form the cores of protein folds, but also promote aggregation. Young genes avoid strongly hydrophobic amino acids, which is presumably the simplest solution to the aggregation problem. Here we show that young genes’ few hydrophobic residues are clustered near one another along the primary sequence, presumably to assist folding. The higher aggregation risk created by the higher hydrophobicity of older genes is counteracted by more subtle effects in the ordering of the amino acids, including a reduction in the clustering of hydrophobic residues until they eventually become more interspersed than if distributed randomly. This interspersion has previously been reported to be a general property of proteins, but here we find that it is restricted to old genes. Quantitatively, the index of dispersion delineates a gradual trend, i.e. a decrease in the clustering of hydrophobic amino acids over billions of years.

scholarly journals Thr176 regulates the activity of the mouse nuclear receptor CAR and is conserved in the NR1I subfamily members PXR and VDR

2005 ◽  
Vol 388 (2) ◽  
pp. 623-630 ◽  
Author(s):  
Akiko UEDA ◽  
Kenji MATSUI ◽  
Yukio YAMAMOTO ◽  
Lars C. PEDERSEN ◽  
Tatsuya SUEYOSHI ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Receptor ◽  
Pregnane X Receptor ◽  
Activation Function ◽  
Constitutive Activity ◽  
Charged Amino Acids ◽  
Binding Surface ◽  
Hydrophobic Amino Acids ◽  
Constitutively Active

The mouse nuclear receptor CAR (constitutively active receptor) is a transcription factor that is activated by phenobarbital-type inducers such as TCPOBOP {1,4 bis[2-(3,5-dichloropyridyloxy)]benzene} in liver in vivo. However, CAR is constitutively active in cell-based transfection assays, the molecular mechanism for which has not been elucidated yet. In the model structure of CAR, Thr176 constitutes a part of the ligand-binding surface, but its side chain is not directed toward the surface, instead it forms a hydrogen bond with Thr350 in the AF2 (activation function 2) domain of CAR. Thr350 is known to regulate CAR activity [Ueda, Kakizaki, Negishi, and Sueyoshi (2002) Mol. Pharmacol. 61, 1284–1288]. Thr176 was mutated to various amino acids to examine whether this interaction played a role in conferring the constitutive activity. Hydrophobic and positively charged amino acids at position 176 abrogated the constitutive activity, whereas polar and negatively charged amino acids retained it. When one of the small hydrophobic amino acids, such as alanine or valine, was substituted for threonine, the mutants were fully activated by TCPOBOP. The co-activator SRC-1 (steroid receptor co-activator-1) regulated the activity changes associated with the mutations. Thr248 and Ser230 are the Thr176-corresponding residues in human pregnane X receptor and mouse vitamin D3 receptor respectively, interacting directly with the conserved threonine in the AF2 domains. Thr248 and Ser230 also regulated the ligand-dependent activity of these receptors by augmenting binding of the receptors to SRC-1. Thr176, Thr248 and Ser230 are conserved residues in the NR1I (nuclear receptor 1I) subfamily members and determine their activity.

Characterization of the hydrophobic properties of amino acids on the basis of their partition and distribution coefficients in the 1-octanol-water system

1991 ◽  
Vol 56 (10) ◽  
pp. 2030-2041 ◽  
Author(s):  
Josef Chmelík ◽  
Jiří Hudeček ◽  
Karol Putyera ◽  
Jiří Makovička ◽  
Vítěz Kalous ◽  
...  
Keyword(s):  
Amino Acids ◽  
Water System ◽  
Distribution Coefficients ◽  
Published Data ◽  
Hydrophobic Properties ◽  
Charged Amino Acids ◽  
Hydrophobic Amino Acids ◽  
Partition Process

The hydrophobic properties of amino acid side chains were characterized on the basis of the partition process in the 1-octanol-water system. The partition coefficients were calculated from the published data and the distribution coefficients were determined experimentally on the basis of a double partition process utilizing the fact that the amino acids pass almost completely into the aqueous phase in the partition process. When the volumes of water and 1-octanol are suitably selected, this fact permits avoidance of the difficulties associated with the determination of amino acids in 1-octanol, where their solubilities are very low. Our scale is the only complete experimental scale based on the partition process of amino acids in the 1-octanol-water system. It follows from comparison of the calculated and the experimental data with the values published for the distribution coefficients of N-acetyl amides of amino acids that the best agreement was achieved for hydrophobic amino acids, while greater differences were observed for hydrophilic amino acids. These differences, expressed as the logarithm of the distribution coefficients, correspond to an average of 0.08 for nonpolar amino acids and 0.30 for acidic and basic amino acids: expressed as relative deviations, these values correspond to 2-10% for nonpolar amino acids, and 5-30% for charged amino acids.

Allosteric inhibition of VIM metallo-β-lactamases by a camelid nanobody

2013 ◽  
Vol 450 (3) ◽  
pp. 477-486 ◽  
Author(s):  
Jean S. Sohier ◽  
Clémentine Laurent ◽  
Andy Chevigné ◽  
Els Pardon ◽  
Vasundara Srinivasan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Active Site ◽  
Substrate Inhibition ◽  
Inhibition Mechanism ◽  
Bacterial Strains ◽  
Single Domain Antibody ◽  
Alanine Scan ◽  
Hydrophobic Amino Acids ◽  
Micromolar Range

MβL (metallo-β-lactamase) enzymes are usually produced by multi-resistant Gram-negative bacterial strains and have spread worldwide. An approach on the basis of phage display was used to select single-domain antibody fragments (VHHs, also called nanobodies) that would inhibit the clinically relevant VIM (Verona integron-encoded MβL)-4 MβL. Out of more than 50 selected nanobodies, only the NbVIM_38 nanobody inhibited VIM-4. The paratope, inhibition mechanism and epitope of the NbVIM_38 nanobody were then characterized. An alanine scan of the NbVIM_38 paratope showed that its binding was driven by hydrophobic amino acids. The inhibitory concentration was in the micromolar range for all β-lactams tested. In addition, the inhibition was found to follow a mixed hyperbolic profile with a predominantly uncompetitive component. Moreover, substrate inhibition was recorded only after nanobody binding. These kinetic data are indicative of a binding site that is distant from the active site. This finding was confirmed by epitope mapping analysis that was performed using peptides, and which identified two stretches of amino acids in the L6 loop and at the end of the α2 helix. Because this binding site is distant from the active site and alters both the substrate binding and catalytic properties of VIM-4, this nanobody can be considered as an allosteric inhibitor.

scholarly journals Probing the specificity of cysteine proteinases at subsites remote from the active site: analysis of P4, P3, P2′ and P3′ variations in extended substrates

2000 ◽  
Vol 347 (1) ◽  
pp. 123-129 ◽  
Author(s):  
Fernada C. Vieira PORTARO ◽  
Ana Beatriz F. SANTOS ◽  
Maria Helena S. CEZARI ◽  
Maria Aparecida JULIANO ◽  
Luiz JULIANO ◽  
...  
Keyword(s):  
Amino Acids ◽  
Significant Influence ◽  
Active Site ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Cysteine Proteinases ◽  
Aminobenzoic Acid ◽  
Site Analysis ◽  
Hydrophobic Amino Acids ◽  
Human Cathepsin

We have determined the kinetic parameters for the hydrolysis by papain, cathepsin B and cathepsin L of internally quenched fluorescent peptides derived from the lead peptides Abz-AAFRSAQ-EDDnp [in which Abz and EDDnp stand for o-aminobenzoic acid and N-(2,4-dinitrophenyl)ethylenediamine respectively], to map the specificity of S4 and S3 subsites, and Abz-AFRSAAQ-EDDnp, to identify the specificity of S2ʹ and S3ʹ. Abz and EDDnp were the fluorescent quencher pair. These two series of peptides were cleaved at the Arg-Ser bond and systematic modifications at P4, P3, P2ʹ and P3ʹ were made. The S4 to S2ʹ subsites had a significant influence on the hydrolytic efficiencies of the three enzymes. Only papain activity was observed to be dependent on S3ʹ, indicating that its binding site is larger than those of cathepsins B and L. Hydrophobic amino acids were accepted at S4, S3, S2ʹ and S3ʹ of the three enzymes. The best substrates for cathepsins L and B had Trp and Asn at P2ʹ respectively; variations at this position were less accepted by these enzymes. The best substrates for papain were peptides containing Trp, Tyr or Asn at P3ʹ. Basic residues at P3 and P4 were well accepted by cathepsin L and papain. We also explored the susceptibility of substrates Abz-AFRSXAQ-EDDnp, modified at P2ʹ (X), to human cathepsin B mutants from which one or two occluding loop contacts had been removed. The modifications at His111 (H111A) and His110 (H110A) of cathepsin B led to an increase in kcat values of one or two orders of magnitude. The hydrolytic efficiencies of these cathepsin B mutants became closer to those of papain or cathepsin L.

scholarly journals Role of conserved hydrophobic amino acids in androgen receptor AF-1 function

2003 ◽  
Vol 31 (3) ◽  
pp. 427-439 ◽  
Author(s):  
R Betney ◽  
IJ McEwan
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Androgen Receptor ◽  
Gene Transcription ◽  
Hydrophobic Residues ◽  
Steroid Receptor Coactivator ◽  
Binding Interface ◽  
Hydrophobic Amino Acids

The intracellular androgen receptor (AR) is a ligand-activated transcription factor. Upon binding the steroids testosterone or dihydrotestosterone, the activated receptor translocates to the nucleus, binds to specific DNA response elements and interacts with the transcription machinery in order to regulate gene transcription. In the present study, we have described a highly conserved region (amino acids 224-258) within the AR AF-1 domain and have investigated the role of conserved bulky hydrophobic residues in gene regulation. Mutating pairs of residues (I229A/L236A; V240A/V242A; L251A/L254A) reduced transactivation activity by 25-40%. Mutating residues M244, L246 and V248 to alanines had a more dramatic affect on receptor activity, disrupting activity by at least 60%. The latter mutations also disrupted binding to the RNA polymerase-associated protein 74 subunit of the general transcription factor TFIIF. The protein conformation and stability of the mutant polypeptide in vitro was not significantly different from the wild type. None of the mutations tested disrupted binding of the AF-1 domain with the coactivator protein steroid receptor coactivator-1a. Thus we have concluded that conserved hydrophobic residues are important for receptor-dependent gene transcription and that M244, L246 and V248 are part of the binding interface for TFIIF.

An Artificial Mitochondrial Tail Signal/Anchor Sequence Confirms a Requirement for Moderate Hydrophobicity for Targeting

2007 ◽  
Vol 27 (6) ◽  
pp. 385-401 ◽  
Author(s):  
Binks W. Wattenberg ◽  
Denise Clark ◽  
Stephanie Brock
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Outer Membrane ◽  
Helical Structure ◽  
Mitochondrial Outer Membrane ◽  
Carboxy Terminus ◽  
Charged Amino Acids ◽  
Hydrophobic Amino Acids ◽  
Signal Anchor ◽  
Anchor Sequence

Tail-anchored proteins are a group of membrane proteins oriented with their amino terminus in the cytoplasm and their carboxy terminus embedded in intracellular membranes. This group includes the apoptosis-mediating proteins of the Bcl-2 family as well as the vesicle targeting proteins of the SNARE group, among others. A stretch of hydrophobic amino acids at the extreme carboxy terminus of these proteins serves both as a membrane anchor and as a targeting signal. Tail-anchored proteins are differentially targeted to either the endoplasmic reticulum or the mitochondrial outer membrane and the mechanism which accomplishes this selective targeting is poorly understood. Here we define important characteristics of the signal/anchor region which directs proteins to the mitochondrial outer membrane. We have created an artificial sequence consisting of a stretch of 16 leucines bounded by positively charged amino acids. Using this template we demonstrate that moderate hydrophobicity distinguishes the mitochondrial tail-anchor sequence from that of the endoplasmic reticulum tail-anchor sequence. A change as small as introduction of a single polar residue into a sequence that otherwise targets to the endoplasmic reticulum can substantially switch targeting to the mitochondrial outer membrane. Further we show that a mitochondrially targeted tail-anchor has a higher propensity for the formation of alpha-helical structure than a sequence directing tail-anchored proteins to the endoplasmic reticulum.

Effects of amino acids on Thiobacillus acidophilus. II. Threonine deaminase

1980 ◽  
Vol 26 (3) ◽  
pp. 385-388 ◽  
Author(s):  
Gérald Proteau ◽  
Marvin Silver
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Ammonium Sulfate ◽  
Active Site ◽  
Binding Sites ◽  
Threonine Deaminase ◽  
Low Concentrations ◽  
High Concentrations ◽  
Thiobacillus Acidophilus ◽  
Optimal Ph

Biosynthetic L-threonine deaminase was partially purified 73-fold with a 60% recovery from Thiobacillus acidophilus by ammonium sulfate fractionation and by Sepharose 6B-C1 chromatography. The optimal pH for enzyme activity was between 9.0 and 10.0 and no optimal pH shift was observed in the presence of L-isoleucine, an inhibitor. The enzyme was effectively inhibited by L-isoleucine and showed homotropic interaction only in the presence of L-isoleucine.Kinetic studies indicate that there are at least two threonine binding sites and at least two isoleucine binding sites. The Km for threonine is 2.5 × 10−3 M. The inhibition due to isoleucine is reversed by low concentrations of L-valine. L-Valine at high concentrations acts as a substrate analogue and competitively inhibits L-threonine binding at the active site; the K1 is 1.6 × 10−2 M.

scholarly journals Polymyxins and quinazolines are LSD1/KDM1A inhibitors with unusual structural features

2016 ◽  
Vol 2 (9) ◽  
pp. e1601017 ◽  
Author(s):  
Valentina Speranzini ◽  
Dante Rotili ◽  
Giuseppe Ciossani ◽  
Simona Pilotto ◽  
Biagina Marrocco ◽  
...  
Keyword(s):  
Amino Acids ◽  
Active Center ◽  
Drug Target ◽  
Malignant Tumors ◽  
Structural Features ◽  
Charged Amino Acids ◽  
Chemistry Research ◽  
Histone Lysine ◽  
Lysine Specific Demethylase ◽  
Positively Charged

Because of its involvement in the progression of several malignant tumors, the histone lysine-specific demethylase 1 (LSD1) has become a prominent drug target in modern medicinal chemistry research. We report on the discovery of two classes of noncovalent inhibitors displaying unique structural features. The antibiotics polymyxins bind at the entrance of the substrate cleft, where their highly charged cyclic moiety interacts with a cluster of positively charged amino acids. The same site is occupied by quinazoline-based compounds, which were found to inhibit the enzyme through a most peculiar mode because they form a pile of five to seven molecules that obstruct access to the active center. These data significantly indicate unpredictable strategies for the development of epigenetic inhibitors.

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