scholarly journals Folding domains and intramolecular ionic interactions of lysine residues in glyceraldehyde 3-phosphate dehydrogenase

1977 ◽  
Vol 161 (1) ◽  
pp. 49-62 ◽  
Author(s):  
J M Lambert ◽  
R N Perham
Keyword(s):  
Catalytic Activity ◽  
Aspartic Acid ◽  
Three Dimensional ◽  
Ion Pair ◽  
Dimensional Structure ◽  
Ionic Interactions ◽  
Rabbit Muscle ◽  
Amino Groups ◽  
Muscle Enzyme ◽  
Lysine Residues

1. Treatment with methyl acetimidate was used to probe the topography of several tetrameric glyceraldehyde 3-phosphate dehydrogenases, in particular the holoenzymes from rabbit muscle and Bacillus stearothermophilus. During the course of the reaction with the rabbit muscle enzyme, the number of amino groups fell rapidly from the starting value of 27 per subunit to a value of approx. five per subunit. This number could be lowered further to values between one and two per subunit by a second treatment with methyl acetimidate. The enzyme remained tetrameric throughout and retained 50% of its initial catalytic activity at the end of the experiment. 2. Use of methyl [1-14C]acetimidate and small-scale methods of protein chemistry showed that only one amino group per subunit, that of lysine-306, was completely unavailable for reaction with imido ester in the native enzyme. This results is consistent with the structure of the highly homologous glyceraldehyde 3-phosphate dehydrogenase of lobster muscle deduced from X-ray-crystallographic analysis, since lysine-306 can be seen to form an intrachain ion-pair with aspartic acid-241 in the hydrophobic environment of a subunit-subunit interface. 3. Several other amino groups in the rabbit muscle enzyme that reacted only slowly with the reagent were also identified chemically. These were found to be located entirely in the C-terminal half of the polypeptides chain, which comprises a folding domain associated with catalytic activity and subunit contact in the three-dimensional structure. Slow reaction of these ‘surface’ amino groups with methyl acetimidate is attributed to intramolecular ionic interactions of the amino groups with neighbouring side-chain carboxyl groups, a conclusion that is compatible with the reported three-dimensional structure and with the dependence of the reaction of ionic stength. 4. Very similar results were obtained with the enzymes from B. stearothermophilus and from ox muscle and ox liver, supporting the view that the ion-pair involving lysine-306 and aspartic acid-241 will be a common structural feature in glyceraldehyde-3-phosphate dehydrogenases. The B. stearothermophilus enzyme was fully active after modification. 5. No differences could be detected between the enzymes from ox muscle and ox liver, in accord with other evidence that points to the identify of these enzymes. 6. The pattern of slowly reacting amino groups in the enzyme from B. stearothermophilus, although similar to that of the mammalian enzymes, indicated one or two additional intramolecular ionic interactions of lysine residues that might contribute to the thermal stability of this enzyme.

scholarly journals Intramolecular ionic interactions of lysine residues and a possible folding domain in fructose diphosphate aldolase

1977 ◽  
Vol 161 (1) ◽  
pp. 63-71 ◽  
Author(s):  
J M Lambert ◽  
R N Perham ◽  
J R Coggins
Keyword(s):  
Primary Structure ◽  
Protein Structures ◽  
Three Dimensional ◽  
Native Enzyme ◽  
Dimensional Structure ◽  
Ionic Interactions ◽  
Rabbit Muscle ◽  
Amino Groups ◽  
Three Dimensional Structure ◽  
Lysine Residues

1. Treatment with methyl acetimidate was used to probe the topography of the tetrameric fructose 1,6-diphosphate aldolase from ox liver. A single treatment with imido ester in the presence or absence of 20mM-fructose 1,6-diphosphate caused the number of amino groups in the enzyme to fall to approx. 30% of the starting number (assumed to be 30 per subunit). The catalytic activity of the aldolase modified in the presence of fructose 1,6-diphosphate was unaffected, whereas that of the enzyme modified in the absence of substrate fell by about 20%. 2. Use of methyl [1-14C]acetimidate and small-scale methods of protein chemistry showed that the amino group of lysine-27 (the numbering is that of the highly homologous rabbit muscle enzyme) is essentially unavailable for amidination in the native enzyme and is therefore predicted to be buried in a hydrophobic environment, probably in the form of an ion-pair with a negatively charged side-chain carboxyl group. All the other lysine residues that reacted poorly with methyl acetimidate in the native enzyme (a total of 7) were found to be within the primary structure bounded by lysine-107 and lysine-227. An important member of this group of lysine residues displaying aberrant reactivity is lysine-227, which is known to form an imine with the substrate as part of the catalytic mechanism of the enzyme. 3. The results of the amidination experiments can be correlated in an interesting way with previous studies of thiol-group modification in the aldolases. Taken together, and arguing in part by analogy with the results of identical experiments with glyceraldehyde 3-phosphate dehydrogenases where the three-dimensional structure is known [Lambert & Perham (1977) Biochem. 4. 161. 49-62], they suggest that the region of primary structure from residues 107-227 may form the whole or part of a three-dimensional structural feature, perhaps a folding domain. A three-dimensional structure deduced from X-ray-crystallographic analysis will be needed to interpret these findings more closely. 4. The amino groups of lysine residues are commonly thought to reside at the ‘surface’ of protein structures. The patterns of specific lysine residues in glyceraldehyde 3-phosphate dehydrogenases and in aldolases that have been found to react poorly with methyl acetimidate in the native enzymes can be attributed to intramolecular ionic interactions deep in hydrophobic pockets and at the protein ‘surface’. Such ionic interactions may contribute significantly to the stability of a given protein.

scholarly journals Chemical cross-linking of a dimeric protein on a modified lectin matrix. A general probe for the chemical topology of oligomeric glycoproteins

1981 ◽  
Vol 193 (3) ◽  
pp. 825-828 ◽  
Author(s):  
S Pillai
Keyword(s):  
Glucose Oxidase ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Cross Linking ◽  
Low Density ◽  
Amino Groups ◽  
Lysine Residues ◽  
The Cross ◽  
Interfacial Surfaces ◽  
Chemical Cross Linking

A dimeric glycoprotein, glucose oxidase, was allowed to react with lysine-specific cross-linkers, both when immobilized on a succinoylated lectin matrix at a critically low density and also at a high density in solution. Analysis of the cross-linked complexes thus obtained led to the following inferences with regard to the structure of this protein. (1) Of the 15 lysine residues on each glucose oxidase protomer, none is available on the non-interfacial surfaces. (2) Assuming that this protein possesses C2 symmetry with isologous bonding between subunits, it may be inferred that on each promoter there are at least two lysine clusters along or close to the interprotomeric interface. (3) These ‘interfacial’ lysine residues on each protomer are so oriented that the epsilon-amino groups of lysine residues a and b on protomer 1 ‘face’, and are very close to, the epsilon-amino groups of lysine residues b' and a' respectively on protomer 2. General inferences on the geometry of dimeric proteins derivable from an analysis of the cross-linked complexes obtained (as well as those not seen) by using this low-density matrix cross-linking approach were enumerated. Modified lectin matrices may prove useful in studying the three-dimensional structure of glycoproteins, particularly non-crystallizable oligomers.

scholarly journals Identification of ‘buried’ lysine residues in two variants of chloramphenicol acetyltransferase specified by R-factors

1981 ◽  
Vol 193 (2) ◽  
pp. 525-539 ◽  
Author(s):  
L C Packman ◽  
W V Shaw
Keyword(s):  
Catalytic Activity ◽  
Quaternary Structure ◽  
Hydrophobic Interactions ◽  
Chloramphenicol Acetyltransferase ◽  
Ion Pair ◽  
Lysine Residue ◽  
Type I ◽  
Amino Groups ◽  
Type Iii ◽  
Lysine Residues

Two variants of chloramphenicol acetyltransferase which are specified by genes on plasmids found in Gram-negative bacteria were subjected to amidination with methyl acetimidate to determine the relative reactivity of surface lysine residues and to search for unreactive or “buried” amino groups which might contribute to stabilization of the native tetramers. Representative examples of the type-I and type-III variants of chloramphenicol acetyltransferase were found to have one lysine residue each in the native state which appears to be inaccessible to methyl acetimidate. The uniquely unreactive residue of the type-I protein is lysine-136, whereas the lysine that is “buried” in the type-III enzyme is provisonally assigned to residue 38 of the prototype sequence. It is suggested that the lysine residue in each case participates in the formation of an ion pair at the intersubunit interface and that the two amino groups in question occupy functionally equivalent positions in the quaternary structures of their respective enzyme variants. Lysine-136 of type-I enzyme is also uniquely unavailable for modification by citraconic anhydride, a reagent used to disrupt the quaternary structure of the native enzyme. Contrary to expectation, exhaustive citraconylation fails to dissociate the tetramer, but does destroy catalytic activity. Removal of citraconyl groups from modified chloramphenicol acetyltransferase is accompanied by a full region of catalytic activity. Analysis of the rate of hydrolysis of citraconyl groups from the modified tetramer by amidination of unblocked amino groups with methyl [14C]acetamidate reveals difference in lability for several of the ten modified lysine residues. Although the unique stability of the quaternary structure of chloramphenicol acetyltransferase may be due to strong hydrophobic interactions, it is argued that lysine-136 may contribute to stability via the formation of an ion pair at the subunit interface.

scholarly journals Crystal structures and hydrogen bonding in the co-crystalline adducts of 3,5-dinitrobenzoic acid with 4-aminosalicylic acid and 2-hydroxy-3-(1H-indol-3-yl)propenoic acid

2014 ◽  
Vol 70 (10) ◽  
pp. 183-187 ◽  
Author(s):  
Graham Smith ◽  
Daniel E. Lynch
Keyword(s):  
Hydrogen Bonding ◽  
Three Dimensional ◽  
Acid Group ◽  
Dimensional Structure ◽  
Aminosalicylic Acid ◽  
Amino Groups ◽  
Propenoic Acid ◽  
Unit Graph ◽  
Ring Centroid ◽  
Hydrogen Bonded

The structures of the co-crystalline adducts of 3,5-dinitrobenzoic acid (3,5-DNBA) with 4-aminosalicylic acid (PASA), the 1:1 partial hydrate, C7H4N2O6·C7H7NO3·0.2H2O, (I), and with 2-hydroxy-3-(1H-indol-3-yl)propenoic acid (HIPA), the 1:1:1d6-dimethyl sulfoxide solvate, C7H4N2O6·C11H9NO3·C2D6OS, (II), are reported. The crystal substructure of (I) comprises two centrosymmetric hydrogen-bondedR22(8) homodimers, one with 3,5-DNBA, the other with PASA, and anR22(8) 3,5-DNBA–PASA heterodimer. In the crystal, inter-unit amine N—H...O and water O—H...O hydrogen bonds generate a three-dimensional supramolecular structure. In (II), the asymmetric unit consists of the three constituent molecules, which form an essentially planar cyclic hydrogen-bonded heterotrimer unit [graph setR32(17)] through carboxyl, hydroxy and amino groups. These units associate across a crystallographic inversion centre through the HIPA carboxylic acid group in anR22(8) hydrogen-bonding association, giving a zero-dimensional structure lying parallel to (100). In both structures, π–π interactions are present [minimum ring-centroid separations = 3.6471 (18) Å in (I) and 3.5819 (10) Å in (II)].

scholarly journals Crystallization and preliminary crystallographic analysis of human muscle phosphofructokinase, the main regulator of glycolysis

2014 ◽  
Vol 70 (5) ◽  
pp. 578-582 ◽  
Author(s):  
Marco Kloos ◽  
Antje Brüser ◽  
Jürgen Kirchberger ◽  
Torsten Schöneberg ◽  
Norbert Sträter
Keyword(s):  
Three Dimensional ◽  
Crystallographic Analysis ◽  
Human Muscle ◽  
Yeast Cells ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Diffusion Method ◽  
Rabbit Muscle ◽  
Main Regulator ◽  
Purification Protocol

Whereas the three-dimensional structure and the structural basis of the allosteric regulation of prokaryotic 6-phosphofructokinases (Pfks) have been studied in great detail, knowledge of the molecular basis of the allosteric behaviour of the far more complex mammalian Pfks is still very limited. The human muscle isozyme was expressed heterologously in yeast cells and purified using a five-step purification protocol. Protein crystals suitable for diffraction experiments were obtained by the vapour-diffusion method. The crystals belonged to space groupP6222 and diffracted to 6.0 Å resolution. The 3.2 Å resolution structure of rabbit muscle Pfk (rmPfk) was placed into the asymmetric unit and optimized by rigid-body and groupB-factor refinement. Interestingly, the tetrameric enzyme dissociated into a dimer, similar to the situation observed in the structure of rmPfk.

scholarly journals Analysis of the Thermostability Determinants of Hyperthermophilic Esterase EstE1 Based on Its Predicted Three-Dimensional Structure

2006 ◽  
Vol 72 (4) ◽  
pp. 3021-3025 ◽  
Author(s):  
Jin-Kyu Rhee ◽  
Do-Yun Kim ◽  
Dae-Gyun Ahn ◽  
Jung-Hyuk Yun ◽  
Seung-Hwan Jang ◽  
...  
Keyword(s):  
Homology Modeling ◽  
Hydrophobic Interactions ◽  
3D Structure ◽  
Three Dimensional ◽  
Ion Pair ◽  
Archaeoglobus Fulgidus ◽  
Dimensional Structure ◽  
Three Dimensional Structure ◽  
Structure Relationship

ABSTRACT The three-dimensional (3D) structure of the hyperthermophilic esterase EstE1 was constructed by homology modeling using Archaeoglobus fulgidus esterase as a reference, and the thermostability-structure relationship was analyzed. Our results verified the predicted 3D structure of EstE1 and identified the ion pair networks and hydrophobic interactions that are critical determinants for the thermostability of EstE1.

Origin of the homochirality of biomolecules

1995 ◽  
Vol 28 (4) ◽  
pp. 473-507 ◽  
Author(s):  
L. Keszthelyi
Keyword(s):  
Amino Acids ◽  
Three Dimensional ◽  
Polarized Light ◽  
Mirror Image ◽  
Reference Points ◽  
Dimensional Structure ◽  
Theoretical Treatment ◽  
Chiral Molecules ◽  
Amino Groups ◽  
The Right

Molecules built up from a given set of atoms may differ in their three-dimensional structure. They may have one or more asymmetric centres that serve as reference points for the steric distribution of the atoms. Carbon atoms, common to all biomolecules, are often such centres. For example, the Cα atom between the carboxyl and amino groups in amino acids is an asymmetric centre: looking ON ward (i.e. from the carbOxyl to the amiNo group, with the Cα oriented so that it is above the carboxyl and amino groups) the radical characterizing the amino acid may be to the right (D-molecules) or to the left (L-molecules). Nineteen of the 20 amino acids occurring in proteins have such a structure (the exception is glycine, where the radical is a hydrogen atom). These pairs of molecules cannot be brought into coincidence with their own mirror image, as is the situation with our hands. The phenomenon has therefore been named handedness, or chirality, from the Greek word cheir, meaning hand. The two forms of the chiral molecules are called enantiomers or antipodes. They differ in rotating the plane of the polarized light either to the right or to the left. The sense of rotation depends on the wavelength of the measuring light, but at a given wavelength it is always opposite for a pair of enantiomers. Chirality may also occur when achiral molecules form chiral substances during crystallization (for example, quartz forms D-quartz or Lquartz). A detailed theoretical treatment of molecular chirality is given by Barron (1991).

scholarly journals The three-dimensional structure of human granzyme B compared to caspase-3, key mediators of cell death with cleavage specificity for aspartic acid in P1

2001 ◽  
Vol 8 (4) ◽  
pp. 357-368 ◽  
Author(s):  
Jennifer Rotonda ◽  
Margarita Garcia-Calvo ◽  
Herb G Bull ◽  
Wayne M Geissler ◽  
Brian M McKeever ◽  
...  
Keyword(s):  
Cell Death ◽  
Aspartic Acid ◽  
Caspase 3 ◽  
Granzyme B ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Three Dimensional Structure ◽  
Cleavage Specificity

The role of disulfide bonds in the conformational stability and catalytic activity of phytase

2004 ◽  
Vol 82 (2) ◽  
pp. 329-334 ◽  
Author(s):  
Xiao-Yun Wang ◽  
Fan-Guo Meng ◽  
Hai-Meng Zhou
Keyword(s):  
Catalytic Activity ◽  
Disulfide Bonds ◽  
Conformational Stability ◽  
Three Dimensional ◽  
Fluorescence Emission ◽  
Dimensional Structure ◽  
Enzyme Activity Assay ◽  
First Order ◽  
Far Ultraviolet

Previous studies have predicted five disulfide bonds in Aspergillus niger phytase (phy A). To investigate the role of disulfide bonds, intrinsic fluorescence spectra, far-ultraviolet circular dichroism (CD) spectra, and an enzyme activity assay were used to compare the differences of catalytic activity and conformational stability of phytase during denaturation in urea in the presence and absence of dithiothreitol (DTT). In the presence of 2 mM DTT, the inactivation and unfolding were greatly enhanced at the same concentration of denaturant. The fluorescence emission maximum red shift and decreases of ellipticity at 222 nm were in accord with the changes of catalytic activity. The kinetics of the unfolding courses were a biphasic process consisting of two first-order reactions in the absence of DTT and a monophasic process of a first-order reaction in the presence of DTT. The results suggested that the loss of enzymatic activity was most likely because of a conformational change, and that disulfide bonds played an important role in three-dimensional structure and catalytic activity.Key words: phytase, urea denaturation, inactivation, disulfide bond.

Polymerization, three-dimensional structure and mechanical properties of Dictyostelium versus rabbit muscle actin filaments

2000 ◽  
Vol 303 (2) ◽  
pp. 171-184 ◽  
Author(s):  
Michel O Steinmetz ◽  
Andreas Hoenger ◽  
Daniel Stoffler ◽  
Angelika A Noegel ◽  
Ueli Aebi ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Rabbit Muscle ◽  
Muscle Actin ◽  
Three Dimensional Structure
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