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 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 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 Acrolein, an irreversible active-site-directed inhibitor of deoxyribose 5-phosphate aldolase?

1976 ◽  
Vol 153 (2) ◽  
pp. 495-497 ◽  
Author(s):  
D C Wilton
Keyword(s):  
Schiff Base ◽  
Rate Constant ◽  
Active Site ◽  
Order Rate Constant ◽  
Lysine Residue ◽  
Prolonged Incubation ◽  
Enzyme Active Site ◽  
First Order ◽  
Substrate Analogue ◽  
Pseudo First Order

The enzyme deoxyribose 5-phosphate aldolase was irreversibly inactivated by the substrate analogue acrolein with a pseudo-first-order rate constant of 0.324 min-1 and a Ki (apparent) of 2.7 × 10(-4) m. No inactivation was observed after prolonged incubation with the epoxide analogues glycidol phosphate and glycidaldehyde. It is suggested that the acrolein is first activated by forming a Schiff base with the enzyme active-site lysine residue and it is the activated inhibitor that reacts with a suitable-active-site nucleophile.

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 Structures of aspartate aminotransferases fromTrypanosoma brucei,Leishmania majorandGiardia lamblia

2015 ◽  
Vol 71 (5) ◽  
pp. 566-571 ◽  
Author(s):  
Jan Abendroth ◽  
Ryan Choi ◽  
Abigail Wall ◽  
Matthew C. Clifton ◽  
Christine M. Lukacs ◽  
...  
Keyword(s):  
Infectious Disease ◽  
Schiff Base ◽  
Trypanosoma Brucei ◽  
Structural Genomics ◽  
Active Site ◽  
Pyridoxal Phosphate ◽  
Inhibitor Design

The structures of three aspartate aminotransferases (AATs) from eukaryotic pathogens were solved within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). Both the open and closed conformations of AAT were observed. Pyridoxal phosphate was bound to the active siteviaa Schiff base to a conserved lysine. An active-site mutant showed thatTrypanosoma bruceiAAT still binds pyridoxal phosphate even in the absence of the tethering lysine. The structures highlight the challenges for the structure-based design of inhibitors targeting the active site, while showing options for inhibitor design targeting the N-terminal arm.

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 The mechanism of action of serine transhydroxymethylase

1970 ◽  
Vol 116 (2) ◽  
pp. 277-286 ◽  
Author(s):  
P. M. Jordan ◽  
M. Akhtar
Keyword(s):  
Schiff Base ◽  
Amino Acid ◽  
Hydrogen Atom ◽  
Mechanism Of Action ◽  
Active Site ◽  
Enzyme Preparation ◽  
Pyridoxal Phosphate ◽  
Acid Oxidase ◽  
Amino Acid Oxidase

1. The preparation of stereospecifically tritiated glycines and the determination of their absolute configurations by the use of d-amino acid oxidase are described. 2. The reaction catalysed by serine transhydroxymethylase, which results in the conversion of glycine into serine, has been separated into at least four partial reactions. It is suggested that the first event in this conversion is the formation of a Schiff base intermediate of glycine and pyridoxal phosphate. The next important step involves the removal of the 2S-hydrogen atom of glycine to give a carbanion intermediate. Experiments pertinent to the mechanism of conversion of this carbanion intermediate into serine are described. 3. The enzyme preparation catalysing the conversion of glycine into serine also participates in the conversion of glycine into threonine and allothreonine. In both these conversions, glycine → serine and glycine → threonine, the 2S-hydrogen atom of glycine is eliminated and the 2R-hydrogen atom of glycine is retained. 4. In the light of these experiments the mechanism of action of serine transhydroxymethylase is discussed. It is suggested that methylenetetrahydrofolate is the carrier of formaldehyde, from which formaldehyde may be liberated at the active site of the enzyme, thus allowing the overall reaction to take place.

An essential arginine residue in the active-site pocket of glycogen phosphorylase

1977 ◽  
Vol 55 (4) ◽  
pp. 465-473 ◽  
Author(s):  
E. C. Y. Li ◽  
R. J. Fletterick ◽  
J. Sygusch ◽  
N. B. Madsen
Keyword(s):  
Amino Acid ◽  
Electron Density ◽  
Active Site ◽  
Protein Molecule ◽  
Competitive Inhibitor ◽  
Arginine Residue ◽  
Sodium Borate ◽  
Appreciable Effect ◽  
First Order ◽  
Active Site Pocket

Phosphorylases a and b (EC 2.4.1.1) were inactivated by selective modification of arginyl residues on reaction with 2,3-butanedione in sodium borate buffer. The rate of inactivation was slightly greater for phosphorylase a than b. The course of inactivation followed pseudo-first-order kinetics with some deviations at low rates or at more than 60% inactivation. The rate of inactivation was first order with respect to butanedione concentration. The inactivation was partially reversible, and ultracentrifugal studies showed no change in subunit association or dissociation. Amino acid analyses indicated that several arginines were modified during inactivation and that no other amino acid was affected. Protection from inactivation was provided by the substrate glucose 1-phosphate (G1P), alone or together with the allosteric activator AMP, as well as by the competitive inhibitor UDP-glucose. The rate of inactivation of phosphorylase b was also retarded by the presence of AMP alone. Glycogen did not have any appreciable effect on inactivation. The Km of G1P for phosphorylase a remained constant over the course of inactivation, while the Km values of G1P and AMP for phosphorylase b increased. The modification of cross-linked tetragonal microcrystals of phosphorylase a followed the same trend as the enzyme in solution, although the rate of inactivation was slower. The X-ray crystallography studies at 6 Å (1 Å = 0.1 nm) resolution, of butanedione-treated cross-linked tetragonal crystals of phosphorylase a showed a large new peak of electron density at the end of a long side chain in the active-site pocket. The substrates G1P and arsenate, as well as UDP-glucose, had previously been shown to bind in that location. Other, small peaks of electron density were found in locations on the outside of the protein molecule. UDP-glucose failed to bind to the active site of crystals which had been treated with butanedione, while AMP, which also binds in the active-site pocket, showed a lower occupancy. This work indicates the presence of a functional arginine residue at the binding site for G1P in glycogen phosphorylases a and b.

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 Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. Binding of pyridoxal phosphate to 5-aminolaevulinate synthetase

1979 ◽  
Vol 177 (2) ◽  
pp. 661-671 ◽  
Author(s):  
R C Davies ◽  
A Neuberger
Keyword(s):  
Schiff Base ◽  
High Activity ◽  
Pyridoxal Phosphate ◽  
Enzymic Activity ◽  
Synthetase Activity ◽  
Dilute Solution ◽  
Concentrated Solutions ◽  
Active Centre ◽  
Rhodopseudomonas Spheroides ◽  
Low Activity

1. Pyridoxal 5′-phosphate is a cofactor essential for the enzymic activity of aminolaevulinate synthetase from Rhodopseudomonas spheroides. It also aids activation of the low-activity enzyme by trisulphides such as cystine trisulphide, whereas inactivation of enzyme is facilitated by its absence. 2. The fluorescence spectrum of purified high-activity enzyme is that expected for a pyridoxal phosphate–Schiff base, but the firmly bound cofactor does not appear to be at the active centre. In dilute solutions of enzyme this grouping is inaccessible to nucleophiles such as glycine, hydroxylamine, borohydride and cyanide, at pH 7.4. 3. An active-centre Schiff base is formed between enzyne and added pyridoxal phosphate, which is accessible to nucleophiles. Concentrated solutions of this enzyme–Schiff base on treatment with glycine yield apo- and semi-apoenzyme, which can re-bind pyridoxal phosphate. 4. Two types of binding of pyridoxal phosphate are distinguishable in dilute solution of enzyme, but these become indistinguishable when concentrated solutions are treated with cofactor. A change occurs in the susceptibility towards borohydride of the fluorescence of the “structural” pyridoxal phosphate. 5. One or two molecules of cofactor are bound per subunit of mol. wt. 50 000 in semiapo- or holo-enzyme. The fluorescence of pyridoxamine phosphate covalently bound to enzyme also indicates one to two nmol of reducible Schiff base per 7000 units of activity in purified and partially purified samples of enzyme. 6. Cyanide does not convert high-activity into low-activity enzyme, but with the enzyme-pyridoxal phosphate complex it forms a yellow fluorescent derivative that is enzymically active.

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