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 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.

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.

Kinetics of volume-sensitive K transport in human erythrocytes: evidence for asymmetry

1989 ◽  
Vol 256 (6) ◽  
pp. C1214-C1223 ◽  
Author(s):  
D. M. Kaji
Keyword(s):  
Statistical Significance ◽  
High Capacity ◽  
Human Erythrocytes ◽  
Kinetic Properties ◽  
Maximal Velocity ◽  
Hill Coefficients ◽  
The Difference ◽  
K Concentration ◽  
The Hill ◽  
External K

The kinetic properties of volume-sensitive K fluxes in swollen human erythrocytes were investigated. Swelling-activated Cl-dependent K influx was a saturable function of external K concentration with a low affinity (apparent Km of 115-130 mM) and high capacity [maximal velocity (Vmax) = 20-30 mmol.l original cells-1.h-1 (mmol.loc-1.h-1)]. The Vmax and apparent Km for Cl-dependent K efflux were lower (Km = 47 mM; Vmax = 2.2 mmol.loc-1.h-1). The Hill coefficients for both K efflux and influx were close to unity, suggesting a single binding site for K. The increase of external K trans-stimulated K efflux, but the increase of intracellular K had no effect on Cl-dependent K influx in swollen cells. Under zero trans conditions, the Vmax (18 vs. 3 mmol.loc-1.h-1) and Km (138 vs. 32) were markedly different for influx and efflux, respectively. These results provide evidence for intrinsic functional asymmetry, such that the transporter is more prevalent and stable in the outward-facing conformation. The mean ratio of Km to Vmax for efflux (12.1) was 1.56 times larger than the same ratio for influx (7.8), but the difference between the means did not reach statistical significance. These kinetic observations are analyzed in terms of the simple carrier and the cotransport models.

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. 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].

Effect of osmotic swelling and shrinkage on Na(+)-K+ pump activity in mammalian cardiac myocytes

1993 ◽  
Vol 265 (5) ◽  
pp. C1201-C1210 ◽  
Author(s):  
D. W. Whalley ◽  
L. C. Hool ◽  
R. E. Ten Eick ◽  
H. H. Rasmussen
Keyword(s):  
Ventricular Myocytes ◽  
Pump Current ◽  
Hill Equation ◽  
Cell Swelling ◽  
Intracellular Na Activity ◽  
Single Ventricular Myocytes ◽  
Hyperosmolar Solutions ◽  
Hill Coefficients ◽  
The Hill ◽  
The Relationship

The effect on the sarcolemmal Na(+)-K+ pump of exposure to anisosmolar solutions was examined using whole cell patch clamping and ion-selective microelectrodes. Na(+)-K+ pump currents were measured in single ventricular myocytes by using pipette Na+ concentrations ([Na]pip) of 0-70 mM. The relationship between [Na]pip and pump current was well described by the Hill equation. The [Na]pip for half-maximal pump current (K0.5) was 21.4 mM in isosmolar (310 mosM) solution. K0.5 was 12.8 mM during cell swelling in hyposmolar solution (240 mosM) and 39.0 mM during cell shrinkage in hyperosmolar solution (464 mosM). The maximal pump currents, derived from the best fit of the Hill equation, and the Hill coefficients were similar in isosmolar, hyposmolar, and hyperosmolar solutions. A sustained (> 20 min) decrease in the intracellular Na+ activity developed during exposure of intact papillary muscles to hyposmolar solutions, and a sustained increase developed during exposure to hyperosmolar solutions. We conclude that osmotic myocyte swelling stimulates the sarcolemmal Na(+)-K+ pump at near-physiological levels of intracellular Na+, whereas shrinkage inhibits the pump. These changes are due to increases and decreases, respectively, in the apparent affinity of the pump for Na+.

scholarly journals Kinetics of rat liver glucokinase. Co-operative interactions with glucose at physiologically significant concentrations

1976 ◽  
Vol 159 (1) ◽  
pp. 7-14 ◽  
Author(s):  
A C Storer ◽  
A Cornish-Bowden
Keyword(s):  
Rat Liver ◽  
Glucose Concentration ◽  
Initial Rate ◽  
Constant Concentration ◽  
Hill Coefficients ◽  
Kinetics Of

The kinetics of glucokinase from rat liver were studied over wide ranges of glucose and MgATP2- concentrations. The initial rate shows a co-operative dependence on the glucose concentration, with Hill coefficients in the range 1.2-1.5. The degree of glucose co-operativity increases with the MgATP2- concentration, but no co-operativity was detected for the dependence of the rate on the MgATP2- concentration. The effects observed occur at physiologically reasonable concentrations of glucose and MgATP2- and are consistent with the presumed function of glucokinase in maintaining a constant concentration of glucose in the blood.

scholarly journals Rates of reactions catalysed by a dimeric enzyme. Effects of the reaction scheme and the kinetic parameters on co-operativity

1991 ◽  
Vol 280 (1) ◽  
pp. 131-137 ◽  
Author(s):  
H Ishikawa ◽  
H Ogino ◽  
H Oshida
Keyword(s):  
Substrate Inhibition ◽  
Reaction Scheme ◽  
Rate Equations ◽  
Kinetic Behaviour ◽  
Hill Coefficients ◽  
S Curve ◽  
Almost All ◽  
The Hill ◽  
Rate Behaviour ◽  
Reaction Schemes

For the reaction S in equilibrium P catalysed by a dimeric enzyme, the reaction schemes are considered on the basis of the KNF model. For each of the ten possible schemes, the rate equation is derived on the basis of the combined steady-state and rapid-equilibrium assumptions. The curves of the plots of initial velocity v versus the substrate concentration [S] and the Hill coefficients h calculated from the rate equations depend strongly on the reaction scheme and the parameter X1. This parameter is defined by log (KS2/KS1) and is a measure of the relative affinities of the first and second protomers for the substrate. When X1 less than 0, v-[S] curves for some schemes exhibit negative co-operativity (h less than 1.0) and v-[S] curves for other schemes are similar to that of the Michaelis-Menten scheme, indicating that, even if there is interaction between the distinct protomers, sigmoidal rate behaviour is not necessarily observed. When X1 greater than 0, all the reaction schemes except one, which shows substrate-inhibition kinetic behaviour, exhibit sigmoidal kinetic behaviour (h greater than 1.0), and at the limit of X1 much greater than 0 the Hill coefficients attain the maximum possible value of 2.0. Furthermore, we have found that, even if X1 = 0, the v-[S] curve for almost all the schemes considered in the present work does not necessarily agree with that for the Michaelis-Menten scheme. This means that the deviation of the v-[S] curve from a hyperbola can be observed even if there is no interaction between the distinct protomers.

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