scholarly journals Effects of lipids on nucleotide inhibition of wheat-germ aspartate transcarbamoylase: evidence of an additional level of control?

1996 ◽  
Vol 313 (2) ◽  
pp. 669-673 ◽  
Author(s):  
Ashan KHAN ◽  
Babur Z. CHOWDHRY ◽  
Robert J. YON
Keyword(s):  
Fatty Acids ◽  
Wheat Germ ◽  
Hill Coefficient ◽  
Aspartate Transcarbamoylase ◽  
Kinetic Effect ◽  
Carbamoyl Phosphate ◽  
Allosteric Mechanism ◽  
Additional Level ◽  
Nucleotide Inhibition ◽  
Metabolic Significance

Wheat-germ aspartate transcarbamoylase, a monofunctional trimer, is strongly inhibited by uridine 5ʹ-monophosphate (UMP), which shows kinetic interactions with the substrate, carbamoyl phosphate, suggesting a classical allosteric mechanism of regulation. Inhibition of the purified enzyme by UMP was amplified in the presence of a variety of ionic lipids at concentrations low enough to preclude denaturation. In the absence of UMP, most of these compounds had no kinetic effect or were slightly activating. Two phospholipids did not show the effect. In a homologous series of fatty acids (C6-C16), the potentiating effect was only seen with homologues greater than C8, reaching a maximum at C12. The effect of dodecanoate (C12) on kinetic cooperativity (UMP as variable ligand) was studied. At each of several fixed concentrations of carbamoyl phosphate, dodecanoate had a pronounced effect on the half-saturating concentration of UMP, which was reduced by about half in every case, indicating substantially tighter binding of UMP. However, dodecanoate had relatively little effect on the kinetic Hill coefficient for the cooperativity of UMP. The possible metabolic significance of these effects is discussed.

scholarly journals Ligand-mediated conformational changes in wheat-germ aspartate transcarbamoylase indicated by proteolytic susceptibility

1984 ◽  
Vol 221 (2) ◽  
pp. 289-296 ◽  
Author(s):  
S C J Cole ◽  
R J Yon
Keyword(s):  
Conformational Changes ◽  
Wheat Germ ◽  
Binding Pocket ◽  
Order Rate Constant ◽  
Aspartate Transcarbamoylase ◽  
Carbamoyl Phosphate ◽  
Peptide Bonds ◽  
First Order ◽  
Mediated Effects ◽  
Arginine Residues

Ligand-mediated effects on the inactivation of pure wheat-germ aspartate transcarbamoylase by trypsin were examined. Inactivation was apparently first-order in all cases, and the effects of ligand concentration on the pseudo-first-order rate constant, k, were studied. Increase in k (labilization) was effected by carbamoyl phosphate, phosphate and the putative transition-state analogue, N-phosphonoacetyl-L-aspartate. Decrease in k (protection) was effected by the end-product inhibitor, UMP, and by the ligand pairs aspartate/phosphate and succinate/carbamoyl phosphate, but not by aspartate or succinate alone up to 10 mM. Except for protection by the latter ligand pairs, all other ligand-mediated effects were also observed on inactivation of the enzyme by Pronase and chymotrypsin. Ligand-mediated effects on the fragmentation of the polypeptide chain by trypsin were examined electrophoretically. Slight labilization of the chain was observed in the presence of carbamoyl phosphate, phosphate and N-phosphonoacetyl-L-aspartate. An extensive protection by UMP was observed, which apparently included all trypsin-sensitive peptide bonds. No significant effect by the ligand pair succinate/carbamoyl phosphate was noted. It is concluded from these observations that UMP triggers an extensive, probably co-operative, transition to a proteinase-resistant conformation, and that carbamoyl phosphate similarly triggers a transition to an alternative, proteinase-sensitive, conformation. These antagonistic conformational changes may account for the regulatory kinetic effects reported elsewhere [Yon (1984) Biochem. J. 221, 281-287]. The protective effect by the ligand pairs aspartate/phosphate and succinate/carbamoyl phosphate, which operates only against trypsin, is concluded to be due to local shielding of essential lysine or arginine residues in the aspartate-binding pocket of the active site, to which aspartate (or its analogue, succinate) can only bind as part of a ternary complex.

scholarly journals Wheat-germ aspartate transcarbamoylase. Kinetic behaviour suggesting an allosteric mechanism of regulation

1972 ◽  
Vol 128 (2) ◽  
pp. 311-320 ◽  
Author(s):  
Robert J. Yon
Keyword(s):  
Wheat Germ ◽  
Initial Rate ◽  
Aspartate Transcarbamoylase ◽  
Kinetic Properties ◽  
Quasi Equilibrium ◽  
Carbamoyl Phosphate ◽  
Partial Explanation ◽  
Enzyme Binding ◽  
Hill Coefficients ◽  
The Hill

1. Some kinetic properties of aspartate transcarbamoylase (EC 2.1.3.2), that had been purified approx. 20-fold from wheat germ, were studied. 2. A plot of enzyme activity against pH showed a low maximum at pH8.4 and a second, higher, maximum at pH10.5. A plot of percentage inhibition by 0.2mm-UMP against pH was approximately parallel to the plot of activity against pH, except that between pH6.5 and 7.5 the enzyme was insensitive to 0.2mm-UMP. 3. Kinetics were studied in detail at pH10.0 and 25°C. In the absence of UMP, initial-rate plots were hyperbolic when the concentration of either substrate was varied. UMP decreased both Vmax. and Km in plots of initial rate against l-aspartate concentration, but the plots remained hyperbolic. However, UMP converted plots of initial rate against carbamoyl phosphate concentration into a sigmoidal shape, without significantly affecting Vmax.. Plots of initial rate against UMP concentration were also sigmoidal. 4. The theoretical model proposed by Monod et al. (1965) gave a partial explanation of these results. When quasi-equilibrium conditions were assumed analysis in terms of this model suggested a trimeric enzyme binding the allosteric ligands, carbamoyl phosphate and UMP, nearly exclusively to the R and T conformational states respectively, and existing predominantly in the R state when ligands were absent. However, the values of the Hill coefficients for the co-operativity of each allosteric ligand were somewhat less than those predicted by the theory. 5. Some of the implications of these results are discussed, and the enzyme is contrasted with the well-known aspartate transcarbamoylase of Escherichia coli.

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.

scholarly journals Inactivation of wheat-germ aspartate transcarbamoylase by the arginine-specific reagent phenylglyoxal

1986 ◽  
Vol 233 (1) ◽  
pp. 303-306 ◽  
Author(s):  
S C Cole ◽  
P A Yaghmaie ◽  
P J Butterworth ◽  
R J Yon
Keyword(s):  
Wheat Germ ◽  
Order Rate Constant ◽  
Bimolecular Reaction ◽  
Arginine Residue ◽  
Aspartate Transcarbamoylase ◽  
Carbamoyl Phosphate ◽  
Active Centre ◽  
First Order ◽  
Degree Of Protection ◽  
Order Rate

Wheat-germ aspartate transcarbamoylase (EC 2.1.3.2) was inactivated by phenylglyoxal in a first-order process, provided that the inactivation time did not exceed 10 min. Apparent first-order rate constants were linearly dependent on phenylglyoxal concentration, indicating a bimolecular reaction between a single active-centre residue and phenylglyoxal, with second-order constant of 0.023 mM-1 X min-1. A plot of apparent first-order rate constant versus pH showed a steep rise above pH 9.5, indicating that the essential residue has a pKa value of 10.5 or higher, consistent with an arginine residue. Saturating concentrations of the following ligands provided a degree of protection (percentages in parentheses) against 1 mM-phenylglyoxal: N-phosphonoacetyl-L-aspartate, a bisubstrate analogue (94%); carbamoyl phosphate (75%); UMP, an end-product inhibitor (53%). Succinate (an analogue of L-aspartate) alone gave no protection, but in combination with carbamoyl phosphate raised the protection to 92%, in agreement with the known binding order of the two substrates. These results indicate that the essential arginine residue is close to the carbamoyl phosphate site, probably oriented towards the aspartate site. Attempts to desensitize the UMP-binding site by reaction with phenylglyoxal, while protecting the active centre, were unsuccessful. The essential active-centre arginine residue is compared with a similar residue in the Escherichia coli enzyme.

scholarly journals Active-site-directed inactivation of wheat-germ aspartate transcarbamoylase by pyridoxal 5′-phosphate

1987 ◽  
Vol 248 (2) ◽  
pp. 403-408 ◽  
Author(s):  
S C J Cole ◽  
R J Yon
Keyword(s):  
Schiff Base ◽  
Active Site ◽  
Wheat Germ ◽  
Pyridoxal Phosphate ◽  
Order Rate Constant ◽  
Competitive Inhibitor ◽  
Aspartate Transcarbamoylase ◽  
Carbamoyl Phosphate ◽  
Active Centre ◽  
First Order

Treatment of 1 microM wheat-germ aspartate transcarbamoylase with 1 mM-pyridoxal 5′-phosphate caused a rapid loss of activity, concomitant with the formation of a Schiff base. Complete loss of activity occurred within 10 min when the Schiff base was reduced with a 100-fold excess of NaBH4. Concomitantly, one amino group per chain was modified. No further residues were modified in the ensuing 30 min. The kinetics of inactivation were examined under conditions where the Schiff base was reduced before assay. Inactivation was apparently first-order. The pseudo-first-order rate constant, kapp., showed a hyperbolic dependence upon the concentration of pyridoxal 5′-phosphate, suggesting that the enzyme first formed a non-covalent complex with the reagent, modification of a lysine then proceeding within this complex. Inactivation of the enzyme by pyridoxal was 20 times slower than that by pyridoxal 5′-phosphate, indicating that the phosphate group was important in forming the initial complex. Partial protection against pyridoxal phosphate was provided by the leading substrate, carbamoyl phosphate, and nearly complete protection was provided by the bisubstrate analogue, N-phosphonoacetyl-L-aspartate, and the ligand-pair carbamoyl phosphate plus succinate. Steady-state kinetic studies, under conditions that minimized inactivation, showed that pyridoxal 5′-phosphate was also a competitive inhibitor with respect to the leading substrate, carbamoyl phosphate. Pyridoxal 5′-phosphate therefore appears to be an active-site-directed reagent. A sample of the enzyme containing one reduced pyridoxyl group per chain was digested with trypsin, and the labelled peptide was isolated and shown to contain a single pyridoxyl-lysine residue. Partial sequencing around the labelled lysine showed little homology with the sequence surrounding lysine-84, an active-centre residue of the catalytic subunit of aspartate transcarbamoylase from Escherichia coli, whose reaction with pyridoxal 5′-phosphate shows many similarities to the results described in the present paper. Arguably the reactive lysine is conserved between the two enzymes whereas the residues immediately surrounding the lysine are not. The same conclusion has been drawn in a comparison of reactive histidine residues in the two enzymes [Cole & Yon (1986) Biochemistry 25, 7168-7174].

scholarly journals Regulatory kinetics of wheat-germ aspartate transcarbamoylase. Adaptation of the concerted model to account for complex kinetic effects of uridine 5′-monophosphate

1984 ◽  
Vol 221 (2) ◽  
pp. 281-287 ◽  
Author(s):  
R J Yon
Keyword(s):  
Wheat Germ ◽  
Initial Rate ◽  
Dissociation Constants ◽  
Phosphate Concentration ◽  
Crude Enzyme ◽  
Aspartate Transcarbamoylase ◽  
Phosphate Binding ◽  
Carbamoyl Phosphate ◽  
Rate Versus ◽  
Kinetic Effects

The kinetic effects of the end-product inhibitor UMP on aspartate transcarbamoylase (EC 2.1.3.2) purified to homogeneity from wheat germ were studied. In agreement with an earlier study of the relatively crude enzyme [Yon (1972) Biochem. J. 128, 311-320], the half-saturating concentrations of UMP and of the first substrate, carbamoyl phosphate (but not of the second, L-aspartate), were found to be strongly interdependent. However, the kinetic behaviour of the pure enzyme differed from that of the crude enzyme in several important respects, namely: (a) the apparent affinity for UMP was lower with the pure enzyme; (b) sigmoidicity was absent from plots of initial rate versus carbamoyl phosphate concentration, each at a fixed UMP concentration; (c) sigmoidicity was greatly exaggerated in plots of initial rate versus UMP concentration, each at a fixed carbamoyl phosphate concentration, owing to the occurrence of a slight but definite maximum in each plot at low UMP concentration; (d) there was a relative increase in this maximum in the presence of N-phosphonacetyl-L-aspartate, an inhibitor competitive with carbamoyl phosphate. It is shown that a modified two-conformation concerted-transition model can be used to account for most of these features of the pure enzyme. The model treats carbamoyl phosphate and UMP as antagonistic allosteric ligands binding to alternative conformational states [Monod, Wyman & Changeux (1965) J. Mol. Biol. 12, 88-118], carbamoyl phosphate binding non-exclusively (dissociation constants 20 microM and 85 microM respectively) and UMP binding exclusively (dissociation constant 2.5 microM). The model postulates further that the conformation with lower affinity for carbamoyl phosphate has the higher value of kcat., and that it binds UMP in competition with carbamoyl phosphate. Parameters giving the best fit of experimental data to this model were found by a non-linear least-squares search procedure.

Fish oil plus wheat germ oil have no prominent effect on homocysteine levels and nutritional indices in hemodialysis patients

2019 ◽  
pp. 1-7
Author(s):  
Hadeer Zakaria ◽  
Tarek M. Mostafa ◽  
Gamal A. El-Azab ◽  
Nagy AH Sayed-Ahmed
Keyword(s):  
Fatty Acids ◽  
Vitamin E ◽  
Fish Oil ◽  
Wheat Germ ◽  
Hemodialysis Patients ◽  
Nutritional Indices ◽  
Omega 3 Fatty Acids ◽  
Omega 3 ◽  
Wheat Germ Oil ◽  
Serum Homocysteine

Abstract. Background: Elevated homocysteine levels and malnutrition are frequently detected in hemodialysis patients and are believed to exacerbate cardiovascular comorbidities. Omega-3 fatty acids have been postulated to lower homocysteine levels by up-regulating metabolic enzymes and improving substrate availability for homocysteine degradation. Additionally, it has been suggested that prevention of folate depletion by vitamin E consumption decreases homocysteine levels. However, data on the effect of omega-3 fatty acids and/or vitamin E on homocysteine levels and nutritional status have been inconclusive. Therefore, this study was planned to examine the effect of combined supplementation of fish oil, as a source of omega-3 fatty acids, with wheat germ oil, as a source of vitamin E, on homocysteine and nutritional indices in hemodialysis patients. Methods: This study was a randomized, double-blind, placebo-controlled trial. Forty-six hemodialysis patients were randomly assigned to two equally-sized groups; a supplemented group who received 3000 mg/day of fish oil [1053 mg omega-3 fatty acids] plus 300 mg/day of wheat germ oil [0.765 mg vitamin E], and a matched placebo group who received placebo capsules for 4 months. Serum homocysteine and different nutritional indices were measured before and after the intervention. Results: Twenty patients in each group completed the study. At the end of the study, there were no significant changes in homocysteine levels and in the nutritional indices neither in the supplemented nor in the placebo-control groups (p > 0.05). Conclusions: Fish oil and wheat germ oil combination did not produce significant effects on serum homocysteine levels and nutritional indices of hemodialysis patients.

scholarly journals Comparative analysis of physiologically important fatty acids

2010 ◽  
pp. 71-76
Author(s):  
Gabriella Kövér ◽  
Zoltán Győri
Keyword(s):  
Fatty Acids ◽  
Fatty Acid Composition ◽  
Fatty Acid ◽  
Comparative Analysis ◽  
Vegetable Oils ◽  
Sunflower Seed ◽  
Wheat Germ ◽  
Grape Seed ◽  
Food Science ◽  
Pumpkin Seed

Fatty acid composition of some vegetable oils, like wheat germ, walnut, peanut, hempseed, linseed, sunflower-seed, olive, rapeseed, grape seed or pumpkin seed, analysed at Food Science Institute of Debrecen University, are summarised here. The effect of heat treatments usually used in Hungarian cuisine was examined in this paper.The influence of different fatty acids on human health is also reviewed.

scholarly journals Quality of wheat germ oil obtained by cold pressing and supercritical carbon dioxide extraction

2013 ◽  
Vol 31 (No. 3) ◽  
pp. 236-240 ◽  
Author(s):  
M.M. Özcan ◽  
A. Rosa ◽  
M.A. Dessi ◽  
B. Marongıu ◽  
A. Pıras ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Wheat Germ ◽  
Alpha Tocopherol ◽  
Fatty Acid Profiles ◽  
Wheat Germ Oil ◽  
Cold Pressing ◽  
Carbon Dioxide Extraction ◽  
Total Fatty Acids ◽  
Cold Pressed

Laboratory-prepared wheat germ oil was obtained by cold pressing and supercritical CO<sub>2</sub> extraction. The main objective was to compare the quality of both oil samples obtained, with emphasis on their fatty acids compositions and tocopherol contents. The percentages of palmitic, oleic, linoleic, and linolenic acids determined in the cold-pressed oil were 15.89, 15.48, 54.88, and 7.34% of total fatty acids, respectively, and those in the oil extracted by supercritical CO<sub>2</sub> were 16.50, 15.05, 54.79, and 7.29% of total fatty acids, respectively. The average proportions of saturated, mono- and polyunsaturated fatty acids calculated for wheat germ oil obtained by cold pressing accounted for 17.15, 17.63, and 62.22% of total fatty acids, respectively, and those calculated for wheat germ oil extracted by supercritical CO<sub>2</sub> were very similar, accounting for 18.14, 17.58, and 62.08% of total fatty acids, respectively. As expected, the fatty acid profiles determined in both oils studied were observed to be almost identical. In contrast, the level of &alpha;-tocopherol in the oil extracted by supercritical CO<sub>2</sub> was found to be considerably higher (1.27 mg/g) than that in the oil obtained by the cold pressing procedure (0.79 mg/g). &nbsp;

scholarly journals Nucleotide ligands protect the inter-domain regions of the multifunctional polypeptide CAD against limited proteolysis, and also stabilize the thermolabile part-reactions of the carbamoyl-phosphate synthase II domains within the CAD polypeptide

1986 ◽  
Vol 236 (2) ◽  
pp. 327-335 ◽  
Author(s):  
E A Carrey
Keyword(s):  
Conformational Stability ◽  
Limited Proteolysis ◽  
Nucleotide Binding ◽  
The Other ◽  
Aspartate Transcarbamoylase ◽  
Biosynthetic Enzymes ◽  
Carbamoyl Phosphate ◽  
Nitrogen Donor ◽  
Phosphate Synthase

Improved methodologies are described which allow the measurement of the part-reactions, with glutamine or ammonia as nitrogen donor, of mammalian carbamoyl-phosphate synthase II (EC 6.3.5.5) through the incorporation of [14C]bicarbonate into either carbamoyl phosphate or carbamoylaspartate. The enzyme is part of the multifunctional polypeptide (CAD) which also comprises the pyrimidine-biosynthetic enzymes aspartate transcarbamoylase (EC 2.1.3.2) and dihydro-orotase (EC 3.5.2.3). The conformational stability of the carbamoyl-phosphate synthase was investigated through the inactivation of the part-reactions which occurred during incubation at 37 degrees C. The domain involved in the removal of the amide N from glutamine was more thermolabile than the ammonia-dependent synthase moiety. The former activity was stabilized in the presence of sodium aspartate or MgATP, whereas the latter was stabilized by MgATP and MgUTP. Binding of MgUTP and MgATP to CAD restricted the initial proteolysis by trypsin and elastase of one or both regions linking the carbamoyl-phosphate synthase domain to the other major domains. A model is described to account for both aspects of nucleotide binding to CAD; these stabilizing effects may be important in the cell, where similar concentrations of nucleotides are found.

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