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 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.

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.

TERATOGENIC EFFECT OF HERBICIDE GLIFONOX IN RATTUS NORVEGICUS, WISTAR STRAIN

2015 ◽  
Vol 8 ◽  
Author(s):  
Ricardo Ortiz Ortega ◽  
Karla S. Martínez Elizalde ◽  
Tomás Ernesto Villamar-Duque
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Rattus Norvegicus ◽  
Biological Material ◽  
Wistar Rats ◽  
Liver Enzymes ◽  
Teratogenic Effect ◽  
Transaminase Activity ◽  
Wistar Strain ◽  
Herbicide Glyphosate

<p>Teratogenic effect of herbicide glyphosate-Roundup, sold under the name Glifotox on Wistar rats was evaluated. The biological material was treated intraperitoneally with glyphosate at concentrations of 100, 125, and 150 mg/kg from gestation day nine. Hysterectomy was performed on day 18 of gestation, and the uterine horns where the embryos were located, in addition to recording the percentage of malformed embryos by modifying the method of Wilson were observed. The liver was removed and quantified by spectrophotometry with transaminase activity showed higher concentrations malformation rate and higher enzyme activity was 125 mg/kg, below is the average of 100 mg/kg and higher concentrations such as 150 mg/kg a large number of resorptions was obtained. It is concluded that glyphosate is toxic affecting the liver and liver enzymes involved in the formation of amino acids also produce delay in embryonic development.</p>

scholarly journals An enzymatic activation of formaldehyde for nucleotide methylation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Charles Bou-Nader ◽  
Frederick W. Stull ◽  
Ludovic Pecqueur ◽  
Philippe Simon ◽  
Vincent Guérineau ◽  
...  
Keyword(s):  
Amino Acids ◽  
Thymidylate Synthase ◽  
Active Site ◽  
De Novo ◽  
Crystallographic Structure ◽  
De Novo Synthesis ◽  
Active Site Residues ◽  
Enzymatic Activation ◽  
Enzyme Cofactors ◽  
Unique Ability

AbstractFolate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.

scholarly journals Sir3p Domains Involved in the Initiation of Telomeric Silencing in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 150 (3) ◽  
pp. 977-986 ◽  
Author(s):  
Yangsuk Park ◽  
John Hanish ◽  
Arthur J Lustig
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Fusion Proteins ◽  
Initiation Site ◽  
Negative Control ◽  
Model System ◽  
Mutant Protein ◽  
Regulatory Domain ◽  
C Terminus ◽  
Necessary And Sufficient

Abstract Previous studies from our laboratory have demonstrated that tethering of Sir3p at the subtelomeric/telomeric junction restores silencing in strains containing Rap1-17p, a mutant protein unable to recruit Sir3p. This tethered silencing assay serves as a model system for the early events that follow recruitment of silencing factors, a process we term initiation. A series of LexA fusion proteins in-frame with various Sir3p fragments were constructed and tested for their ability to support tethered silencing. Interestingly, a region comprising only the C-terminal 144 amino acids, termed the C-terminal domain (CTD), is both necessary and sufficient for restoration of silencing. Curiously, the LexA-Sir3N205 mutant protein overcomes the requirement for the CTD, possibly by unmasking a cryptic initiation site. A second domain spanning amino acids 481-835, termed the nonessential for initiation domain (NID), is dispensable for the Sir3p function in initiation, but is required for the recruitment of the Sir4p C terminus. In addition, in the absence of the N-terminal 481 amino acids, the NID negatively influences CTD activity. This suggests the presence of a third region, consisting of the N-terminal half (1-481) of Sir3p, termed the positive regulatory domain (PRD), which is required to initiate silencing in the presence of the NID. These data suggest that the CTD “active” site is under both positive and negative control mediated by multiple Sir3p domains.

Amino acids flanking the central core of Cu,Zn superoxide dismutase are important in retaining enzyme activity after autoclaving

2016 ◽  
Vol 34 (3) ◽  
pp. 475-485 ◽  
Author(s):  
Arun Kumar ◽  
Vinay Randhawa ◽  
Vishal Acharya ◽  
Kashmir Singh ◽  
Sanjay Kumar
Keyword(s):  
Amino Acids ◽  
Superoxide Dismutase ◽  
Enzyme Activity ◽  
Central Core

scholarly journals Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

1987 ◽  
Vol 7 (1) ◽  
pp. 294-304 ◽  
Author(s):  
D Pilgrim ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Proteolytic Processing ◽  
Mitochondrial Matrix ◽  
Wild Type ◽  
Mature Form ◽  
Amino Terminal ◽  
The Matrix

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.

A possible role of arginase in the regulation of polyamine biosynthesis in the rat thyroid

1981 ◽  
Vol 98 (1) ◽  
pp. 57-61 ◽  
Author(s):  
Shigeru Matsuzaki ◽  
Mitsuo Suzuki ◽  
Koei Hamana
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Arginase Activity ◽  
Polyamine Biosynthesis ◽  
Rat Thyroid ◽  
Arginine Concentration

Abstract. Effect of chronic methylthiouracil (MTU) treatment on the thyroid arginase activity and thyroidal concentration of arginine, ornithine and other amino acids was studied in the rat. The activity of thyroid arginase increased significantly at 15 days of MTU treatment and the elevated enzyme activity was reduced to normal by l-thyroxine (T4) injection. The thyroidal concentration of polyamines was increased by MTU and decreased by T4 with the exception of spermine. The thyroidal concentration of arginine and lysine, a substrate and an inhibitor for arginase respectively decreased significantly, while that of ornithine remained unchanged after MTU treatment. T4 injected to MTU-pretreated rats restored the decreased arginine concentration to normal. These results suggest that ornithine supply for polyamine biosynthesis is regulated by the level of both arginase and lysine in the thyroid.

Active amino acids of the Kell blood group protein and model of the ectodomain based on the structure of neutral endopeptidase 24.11

Blood ◽  
2003 ◽  
Vol 102 (8) ◽  
pp. 3028-3034 ◽  
Author(s):  
Soohee Lee ◽  
Asim K. Debnath ◽  
Colvin M. Redman
Keyword(s):  
Amino Acids ◽  
Blood Group ◽  
Active Site ◽  
Neutral Endopeptidase ◽  
Site Directed Mutagenesis ◽  
Peptide Hydrolysis ◽  
Enzyme Active Site ◽  
Endopeptidase 24.11 ◽  
Outer Domain ◽  
Group Protein

Abstract In addition to its importance in transfusion, Kell protein is a member of the M13 family of zinc endopeptidases and functions as an endothelin-3–converting enzyme. To obtain information on the structure of Kell protein we built a model based on the crystal structure of the ectodomain of neutral endopeptidase 24.11 (NEP). Similar to NEP, the Kell protein has 2 globular domains consisting mostly of α-helical segments. The domain situated closest to the membrane contains both the N- and C-terminal sequences and the enzyme-active site. The outer domain contains all of the amino acids whose substitutions lead to different Kell blood group phenotypes. In the model, the zinc peptidase inhibitor, phosphoramidon, was docked in the active site. Site-directed mutagenesis of amino acids in the active site was performed and the enzymatic activities of expressed mutant Kell proteins analyzed and compared with NEP. Our studies indicate that Kell and NEP use the same homologous amino acids in the coordination of zinc and in peptide hydrolysis. However, Kell uses different amino acids than NEP in substrate binding and appears to have more flexibility in the composition of amino acids allowed in the active site.

Steroids and steroidases. IX. Activation parameters and the mechanism of base- and enzyme-catalyzed isomerizations of androst-5-ene-3,17-dione. The nature of the active center of the Δ5 → Δ4-3-ketosteroid isomerase of Pseudomonas testosteroni

1969 ◽  
Vol 47 (23) ◽  
pp. 4459-4466 ◽  
Author(s):  
J. Bryan Jones ◽  
Donald C. Wigfield
Keyword(s):  
Amino Acids ◽  
Active Center ◽  
Activation Parameters ◽  
Circumstantial Evidence ◽  
Kcal Mole ◽  
Acid Base ◽  
Ketosteroid Isomerase ◽  
Enzyme Action ◽  
Enthalpy Of Activation

Determination of the activation parameters for the acid-, base-, and enzyme-catalyzed isomerizations of androst-5-ene-3,17-dione has revealed that the facility of the enzymic process is mainly due to an extremely low enthalpy of activation of 5.0 kcal mole−1. Further circumstantial evidence regarding the nature of the reacting groups at the active center has also been obtained, and a mechanism of enzyme action is proposed employing tyrosine and histidine as the principal amino acids responsible for catalyzing the isomerization.

Sign in / Sign up

Export Citation Format

Share Document