Structure and function of aspartate transcarbamoylase studied using chymotrypsin as a probe

1978 ◽  
Vol 56 (6) ◽  
pp. 654-658 ◽  
Author(s):  
William W.-C. Chan ◽  
Caroline A. Enns
Keyword(s):  
Active Sites ◽  
Sodium Dodecyl ◽  
Peptide Bond ◽  
Structure And Function ◽  
Catalytic Subunit ◽  
Native Enzyme ◽  
Aspartate Transcarbamoylase ◽  
Carbamoyl Phosphate ◽  
And Function ◽  
Extended Exposure

Aspartate transcarbamoylase from Escherichia coli is composed of six catalytic (c) and six regulatory (r) polypeptides. We have studied the structure and function of this enzyme using chymotrypsin as a probe. The protease inactivates the isolated catalytic subunit (c3) but has no effects on the native enzyme (c6r6). Under identical conditions, the c3r6 complex is inactivated at a much slower rate than c3. The presence of the substrate analogue succinate together with carbamoyl phosphate reduces substantially the rate of inactivation. Extended exposure to chymotrypsin converts the catalytic subunit into a partially active derivative with a fourfold higher Michaelis constant. This derivative is indistinguishable from the unmodified catalytic subunit in gel electrophoresis under nondenaturing conditions. However, in the presence of sodium dodecyl sulfate, the major fragment in the electropherogram is smaller than that of the intact catalytic polypeptide. The results could be explained by postulating the presence of a chymotrypsin-sensitive peptide bond at or near the active site. Since X-ray crystallographic studies have indicated that the active sites are located in a central cavity, the resistance of the native enzyme towards inactivation may be due to the inability of chymotrypsin to enter this cavity.

Aspartate transcarbamoylase: loss of homotropic but not heterotropic interactions upon modification of the catalytic subunit with a bifunctional reagent

1979 ◽  
Vol 57 (6) ◽  
pp. 798-805 ◽  
Author(s):  
William W.-C. Chan ◽  
Caroline A. Enns
Keyword(s):  
Conformational Changes ◽  
Substrate Binding ◽  
Catalytic Subunit ◽  
Native Enzyme ◽  
Aspartate Transcarbamoylase ◽  
Substrate Affinity ◽  
Carbamoyl Phosphate ◽  
Catalytic Subunits ◽  
Regulatory Subunits ◽  
Michaelis Constants

The role of conformational changes in the allosteric mechanism of aspartate transcarbamoylase from Escherichia coli was studied by reacting the isolated catalytic subunit with the bifunctional reagent tartryl diazide. Two derivatives differing moderately in substrate affinity were obtained depending on whether the reaction was conducted in the presence or absence of the substrate analogue succinate and carbamoyl phosphate. The modification was not accompanied by aggregation or dissociation. The modified catalytic subunits retained the ability to reassociate with unmodified regulatory subunits and produced hybrids similar in size to the native enzyme. These hybrids were appreciably sensitive to the allosteric effectors ATP and CTP but unlike native enzyme showed no cooperativity in substrate binding. The Michaelis constants of these hybrids for aspartate were intermediate between that of the isolated catalytic subunit and that of the relaxed state. Activation by ATP was caused by a reduction in Km to the value characteristic of the relaxed state whereas CTP inhibited by lowering the Vmax. The properties of the hybrids are strikingly similar to the modified enzyme obtained by Kerbiriou and Hervé from cells grown in the presence of 2-thiouracil. However, the crucial modifications are found in the regulatory subunits of the enzyme studied by these authors whereas they are located in the catalytic subunits of the hybrids reported here. Our results suggest that interactions between the catalytic and regulatory subunits have considerable effects on the state of the substrate binding sites in the native enzyme.

scholarly journals Local frustration around enzyme active sites

2019 ◽  
Vol 116 (10) ◽  
pp. 4037-4043 ◽  
Author(s):  
Maria I. Freiberger ◽  
A. Brenda Guzovsky ◽  
Peter G. Wolynes ◽  
R. Gonzalo Parra ◽  
Diego U. Ferreiro
Keyword(s):  
Primary Structure ◽  
Active Sites ◽  
Protein Sequences ◽  
Structure And Function ◽  
Energy Landscapes ◽  
Biological Functions ◽  
Complex Energy ◽  
Oligomeric State ◽  
Catalytic Sites ◽  
And Function

Conflicting biological goals often meet in the specification of protein sequences for structure and function. Overall, strong energetic conflicts are minimized in folded native states according to the principle of minimal frustration, so that a sequence can spontaneously fold, but local violations of this principle open up the possibility to encode the complex energy landscapes that are required for active biological functions. We survey the local energetic frustration patterns of all protein enzymes with known structures and experimentally annotated catalytic residues. In agreement with previous hypotheses, the catalytic sites themselves are often highly frustrated regardless of the protein oligomeric state, overall topology, and enzymatic class. At the same time a secondary shell of more weakly frustrated interactions surrounds the catalytic site itself. We evaluate the conservation of these energetic signatures in various family members of major enzyme classes, showing that local frustration is evolutionarily more conserved than the primary structure itself.

scholarly journals Tungstoenzymes: Occurrence, Catalytic Diversity and Cofactor Synthesis

Inorganics ◽  
2020 ◽  
Vol 8 (8) ◽  
pp. 44
Author(s):  
Carola S. Seelmann ◽  
Max Willistein ◽  
Johann Heider ◽  
Matthias Boll
Keyword(s):  
Abc Transporter ◽  
Aromatic Compounds ◽  
Metal Binding ◽  
Active Sites ◽  
Structure And Function ◽  
Hydration Reaction ◽  
Dimethyl Sulfoxide Reductase ◽  
Reduction Of Co2 ◽  
And Function ◽  
Electron Transfers

Tungsten is the heaviest element used in biological systems. It occurs in the active sites of several bacterial or archaeal enzymes and is ligated to an organic cofactor (metallopterin or metal binding pterin; MPT) which is referred to as tungsten cofactor (Wco). Wco-containing enzymes are found in the dimethyl sulfoxide reductase (DMSOR) and the aldehyde:ferredoxin oxidoreductase (AOR) families of MPT-containing enzymes. Some depend on Wco, such as aldehyde oxidoreductases (AORs), class II benzoyl-CoA reductases (BCRs) and acetylene hydratases (AHs), whereas others may incorporate either Wco or molybdenum cofactor (Moco), such as formate dehydrogenases, formylmethanofuran dehydrogenases or nitrate reductases. The obligately tungsten-dependent enzymes catalyze rather unusual reactions such as ones with extremely low-potential electron transfers (AOR, BCR) or an unusual hydration reaction (AH). In recent years, insights into the structure and function of many tungstoenzymes have been obtained. Though specific and unspecific ABC transporter uptake systems have been described for tungstate and molybdate, only little is known about further discriminative steps in Moco and Wco biosynthesis. In bacteria producing Moco- and Wco-containing enzymes simultaneously, paralogous isoforms of the metal insertase MoeA may be specifically involved in the molybdenum- and tungsten-insertion into MPT, and in targeting Moco or Wco to their respective apo-enzymes. Wco-containing enzymes are of emerging biotechnological interest for a number of applications such as the biocatalytic reduction of CO2, carboxylic acids and aromatic compounds, or the conversion of acetylene to acetaldehyde.

scholarly journals Paramyosin in invertebrate muscles. II. Content in relation to structure and function.

1976 ◽  
Vol 71 (1) ◽  
pp. 273-279 ◽  
Author(s):  
R J Levine ◽  
M Elfvin ◽  
M M Dewey ◽  
B Walcott
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Thick Filament ◽  
Structure And Function ◽  
Active Tension ◽  
Tension Development ◽  
Force Development ◽  
And Function

By quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, paramyosin:myosin heavy chain molecular ratios were calculated for three molluscan muscles:Aequipecten striated adductor, Mercenaria opaque adductor, and Mytilus anterior byssus retractor; and four arthropodan muscles:Limulus telson, Homarus slow claw. Balanus scutal depressor, and Lethocerus air tube retractor. These ratios correlate positively with both thick filament dimensions and maximum active tension development in these tissues. The role of paramyosin in these muscles is discussed with respect to the following characteristics: force development, "catch," and extreme reversible changes in length.

scholarly journals Significance of arginine radicals for sturgeon gonadotropin secondary structure and function

2010 ◽  
Vol 64 (3-4) ◽  
pp. 144-148
Author(s):  
Henriks Zenkevičs ◽  
Ilze Vosekalna ◽  
Vija Vose
Keyword(s):  
Secondary Structure ◽  
Active Sites ◽  
Positive Charge ◽  
Cd Spectroscopy ◽  
Structure And Function ◽  
Dimeric Molecule ◽  
Gonadotropic Hormone ◽  
Chemically Modified ◽  
Individual Subunit ◽  
And Function

Significance of arginine radicals for sturgeon gonadotropin secondary structure and function Guanidine groups of arginine side chains were selectively chemically modified with 1,2-cyclohexanedione (CHD) in sturgeon (Acipenser güldenstädti Br.) gonadotropic hormone (GTH) and in its subunits. It was found that only two of the six guanidines were accessible for the reagent and each of the two modified groups was bound to an individual subunit. The results showed that both modified groups were located on the surface of the hormone dimeric molecule. CD-spectroscopy of the modified hormonal preparations did not indicate any considerable changes in their secondary structure. On the basis of the data obtained, a conclusion was made that the free guanidine groups are of exclusive importance for the hormone function at the receptor level as the bearers of the positive charge in the functionally important active sites or effector zones located on the surface of the hormone molecule. Also, it was shown that the guanidine groups played a certain role in sustaining the functionally effective spatial structure of the subunits and GTH.

scholarly journals Deglycosylation of glycoproteins with trifluoromethanesulphonic acid: elucidation of molecular structure and function

2003 ◽  
Vol 376 (2) ◽  
pp. 339-350 ◽  
Author(s):  
Albert S. B. EDGE
Keyword(s):  
Molecular Structure ◽  
Peptide Bond ◽  
Structure And Function ◽  
Glycosidic Linkage ◽  
Biological Functions ◽  
Peptide Bonds ◽  
Post Translational Modifications ◽  
And Function ◽  
Carbohydrate Chains ◽  
Molecular Structure And Function

The alteration of proteins by post-translational modifications, including phosphorylation, sulphation, processing by proteolysis, lipid attachment and glycosylation, gives rise to a broad range of molecules that can have an identical underlying protein core. An understanding of glycosylation of proteins is important in clarifying the nature of the numerous variants observed and in determining the biological roles of these modifications. Deglycosylation with TFMS (trifluoromethanesulphonic acid) [Edge, Faltynek, Hof, Reichert, and Weber, (1981) Anal. Biochem. 118, 131–137] has been used extensively to remove carbohydrate from glycoproteins, while leaving the protein backbone intact. Glycosylated proteins from animals, plants, fungi and bacteria have been deglycosylated with TFMS, and the most extensively studied types of carbohydrate chains in mammals, the N-linked, O-linked and glycosaminoglycan chains, are all removed by this procedure. The method is based on the finding that linkages between sugars are sensitive to cleavage by TFMS, whereas the peptide bond is stable and is not broken, even with prolonged deglycosylation. The relative susceptibility of individual sugars in glycosidic linkage varies with the substituents at C-2 and the occurrence of amido and acetyl groups, but even the most stable sugars are removed under conditions that are sufficiently mild to prevent scission of peptide bonds. The post-translational modifications of proteins have been shown to be required for diverse biological functions, and selective procedures to remove these modifications play an important role in the elucidation of protein structure and function.

scholarly journals Wheat-germ aspartate transcarbamoylase. The effect of ligands on the inactivation of the enzyme by trypsin and denaturing agents

1973 ◽  
Vol 131 (4) ◽  
pp. 699-706 ◽  
Author(s):  
Robert J. Yon
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Wheat Germ ◽  
Sodium Dodecyl ◽  
Aspartate Transcarbamoylase ◽  
Carbamoyl Phosphate ◽  
Low Concentrations ◽  
Alkaline Conditions ◽  
High Concentrations ◽  
Very High ◽  
Resistant State

In the absence of added ligands aspartate transcarbamoylase (EC 2.1.3.2) from wheat germ is inactivated fairly rapidly by trypsin, by heat (60°C), by highly alkaline conditions (pH11.3) and by sodium dodecyl sulphate. Addition of UMP alone, at low concentrations, decreases the rate of inactivation by each of these agents significantly. Carbamoyl phosphate alone does not alter the rate of inactivation by trypsin and by the detergent, but it antagonizes the effect of UMP in protecting the enzyme against these agents. These results have been interpreted to mean that two conformational states are reversibly accessible to the enzyme, namely an easily inactivated state favoured in the presence of carbamoyl phosphate and a more resistant state favoured in the presence of UMP. In the absence of ligands the enzyme is in the easily inactivated conformation. At very high concentrations l-aspartate also protects the enzyme but to a smaller extent than UMP. Some implications of these results are discussed.

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