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 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 The reaction of amino-oxyacetate with pyridoxal phosphate-dependent enzymes

1978 ◽  
Vol 171 (3) ◽  
pp. 771-779 ◽  
Author(s):  
R A John ◽  
A Charteris
Keyword(s):  
Enzyme Activity ◽  
Pyridoxal Phosphate ◽  
Equilibrium Constants ◽  
Order Rate Constant ◽  
Bimolecular Reaction ◽  
Dopa Decarboxylase ◽  
Aspartate Transaminase ◽  
First Order ◽  
Order Rate ◽  
Kinetics Of

The carbonyl reagent amino-oxyacetate is frequently used in metabolic studies to inhibit individual pyridoxal phosphate enzymes. The reaction of this compound with three such enzymes, aspartate transaminase, 4-aminobutyrate transaminase and dopa (3,4-dihydroxyphenylalanine) decarboxylase, was studied to determine the extent to which the inhibition is reversible and the rates at which it takes place. Reactions were followed by observing changes in the absorption spectra of the bound coenzyme and by measuring loss of enzyme activity. The reactions with aspartate transaminase and aminobutyrate transaminase were not rapidly reversible and had second-order rate constants (21 degrees C) of 400 M-1.s.1 and 1300 M-1.s-1 respectively and all all concentrations studied showed the kinetics of a simple bimolecular reaction. The reaction with 4-aminobutyrate transaminase could not be reversed and that with aspartate transaminase could only be reversed significantly by addition of cysteinesulphinate to convert the enzyme into its pyridoxamine form. The first-order rate constant (21 degrees C) for the reverse reaction was 4 × 10(-5)s-1. Dopa decarboxylase inhibition by amino-oxyacetate was more rapid and more readily reversible, but measurements of rate and equilibrium constants were not obtained for this enzyme.

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 Compounds of 6, 13-Diamino-6, 13-Dimethyl-1, 4, 8, 11 - Tetraazacyclotetradecane

2021 ◽  
Author(s):  
◽  
Asokamali Siriwardena
Keyword(s):  
Rate Constants ◽  
Magnetic Measurements ◽  
Order Rate Constant ◽  
Acidic Solutions ◽  
First Order ◽  
Order Rate ◽  
Pendant Arm ◽  
Nickel Copper ◽  
13C Nuclear Magnetic Resonance ◽  
Kinetics Of

<p>The reaction of bis-(diaminoethane)nickel(II) chloride, ([Ni(en)2]Cl2 in methanol with formaldehyde and nitroethane in the presence of triethylamine proceeds readily to produce (6, 13-dimethyl-6, 13-dinitro-1, 4, 8, 11-tetraazacyclotetradecane)nickel(II) chloride, [Ni(dini)] - Cl2. Reduction of the nitro groups of this compound by catalytic hydrogenation yields three isomers of the pendant arm macrocyclic complex (6, 13-diamino-6, 13-dimethyl-1, 4, 8, 11-tetraazachyclotetradecane)nickel(II) chloride, designated a-, b- and c-[Ni(diam)]Cl2. These were separated by fractional crystallization. The aisomer was observed to isomerizes slowly in solution to the b- form. A parallel dissociation reaction of the a- isomer was also observed. The demetallation of a- and b- isomers of the diam complex of nickel by reaction with cyanide or concentrated acid at 140 degrees C produces the macrocycle meso-(6, 13-diamino-6, 13-dimethyl-1, 4, 8, 11-tetraazacyclotetra-decane), diam. A variety of hexamine, pentamine and tetramine complexes of diam with nickel(II), copper(II), cobalt(II) and (III), chromium(III), palladium(II), rhodium(III), zinc(II) and cadmium(II) were prepared. Hexamine and tetramine forms of labile metal complexes could be rapidly and reversibly interconverted by altering the pH. The hexamine cobalt(III) cation, [Co(diam)]3+ was by far the most inert of the prepared cobalt(III) complexes, remaining unaffected in hot acidic solutions. In contrast, a single pendant arm of the hexamine [Cr(diam)]3+ cation could be dissociated in acid. (Two possibly triamine complexes of lead were also prepared). These compounds were characterized by elemental analysis, magnetic measurements, electronic, infrared, 1H and 13C nuclear magnetic resonance spectra. The pendant arm protonation constants (log K) of diam and selected complexes of nickel, copper and palladium were calculated from potentiometric titration measurements at 25 degrees C. The log K values for diam at 25 degrees C (I = 0.1 M NaclO4) were 11.15, 9.7, 6.2 and 5.3. Kinetics of the parallel isomerization and dissociation of a-[Ni(dimH2)]4+ in HCl/NaCl solutions were monitored spectrophotometrically at 50 degrees C. The rate of reaction in acidic solutions showed a non-linear dependency on acid concentration. The observed first order rate constant (kobs) for disappearance of a-[Ni(diamH2)]4+ (by isomerization and dissociation) in 2.0 M HCl, 0.1 M NaOH and 2.0 M NaCl were 3.05 x 10-4, 2.0(3) x 10-2 and 5.0 x 10-5 s-1 respectively. The rate of the dissociation component of the reaction of a-[Ni(diamH2)]4+ in 2.0 M HCl at 50 degrees C was 1.82 x 10-7 s-1. Acid bydrolysis kinetics of (Cu[diamH2])(ClO4)4 in hydrochloric acid and perchloric acid at 50 and 70 degrees C were studied spectrophotometrically. The reactions were slow and the observed first order rate constants were to a first approximation independent of the particular acid or its concentration. The observed first order rate constants were 1 x 10-9 and 8 x 10-9 s-1 at 50 and 70 degrees C respectively. Questions about the nature of the reaction being followed have been raised.</p>

A method of determining electrical potential gradient across mitochondrial membrane in perfused rat hearts

1993 ◽  
Vol 265 (2) ◽  
pp. H445-H452 ◽  
Author(s):  
B. Wan ◽  
C. Doumen ◽  
J. Duszynski ◽  
G. Salama ◽  
K. F. LaNoue
Keyword(s):  
Mitochondrial Membrane ◽  
Electrical Potential ◽  
Potential Gradient ◽  
Order Rate Constant ◽  
Isolated Rat Heart ◽  
First Order ◽  
Order Rate ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Electrical Potential Gradient

The electrical potential gradient across the mitochondrial membrane (delta psi m) in perfused rat hearts was estimated by calculating the equilibrium distribution of the lipophilic cation tetraphenylphosphonium (TPP+), using measured kinetic constants of uptake and release of TPP+. First-order rate constants of TPP+ uptake were measured during 30-min perfusions of intact rat hearts with tracer amounts (5.0 nM) of tritium-labeled TPP+ ([3H]TPP+) in the perfusate. This was followed by a 30-min washout, during which the first-order rate constant of efflux was estimated. Values of [3H]TPP+ outside the heart and total [3H]TPP+ inside the heart at equilibrium were calculated. From this information and separately estimated time-averaged plasma membrane potentials (delta psi c) it was possible to calculate free cytosolic [3H]TPP+ at equilibrium. It was also possible to calculate free intramitochondrial [3H]TPP+ at equilibrium as the difference between total tissue [3H]TPP+ minus free cytosolic TPP+ and the sum of all the bound [3H]TPP+. Bound [3H]TPP+ was determined from [3H]TPP+ binding constants measured in separate experiments, using both isolated mitochondria and isolated cardiac myocytes under conditions where both delta psi m and delta psi c were zero. Delta psi m was calculated from the intramitochondrial and cytosolic free TPP+ concentrations using the Nernst equation. Values of delta psi m were 144.9 +/- 2.0 mV in hearts perfused with 5 mM pyruvate and 118.2 +/- 1.4 mV in hearts perfused with 11 mM glucose, in good agreement with delta psi m obtained from isolated rat heart mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

The Dephosphorylation of Adenosine 5′ -Triphosphate in a Binary and Ternary Zn2+ Complex

1974 ◽  
Vol 29 (11-12) ◽  
pp. 680-682 ◽  
Author(s):  
Peter Amsler ◽  
David Buisson ◽  
Helmut Sigel
Keyword(s):  
Rate Constant ◽  
Ternary Complex ◽  
Order Rate Constant ◽  
Reactive Species ◽  
Ph Optimum ◽  
First Order ◽  
Order Rate

The dephosphorylation of ATP was characterized by determining the dependence of the first-order rate constant on pH in the absence and presence of Zn2+ and together with Zn2+ and 2,2′-bipyridyl. The Zn2+-accelerated reaction passes through a pH optimum at about 8. The decrease in the rate at higher pH is due to the formation of Zn(ATP) (OH)3-; this species is relatively insensitive towards dephosphorylation. It is concluded that Zn(ATP)2- is the reactive species and that the interaction between N (7) and Zn2+ in this complex is crucial for its reactivity. In the presence of 2,2′-bipyridyl (Bipy) the ternary complex, Zn (Bipy) (ATP)2-, is formed which is rather stable towards dephosphorylation. It is suggested that the described effects of acceleration and inhibition are helpful for understanding the recycled processes in nature.

HUMAN MEIZOTHROMBIN 1, ITS ISOLATION AND PROPERTIES

1987 ◽  
Author(s):  
Zbigniew S Latallo ◽  
Craig M Jackson
Keyword(s):  
Rate Constants ◽  
Antithrombin Iii ◽  
Order Rate Constant ◽  
Red Cross ◽  
Apparent Lack ◽  
First Order ◽  
Order Rate ◽  
Matching Grant ◽  
Echis Carinatus

Meizothrombin (MT) and meizothrombin des Fragment 1 (MT1) are intermediates in the conversion of prothrombin to α-thrombin (αTH). Due to their transient character, properties of these enzymes are difficult to establish. Isolation of MT1 was achieved by affinity chromatography on D-Phe-Pro-Arginal (FPRal)immobilized on Affi-Gel 10 as originally employed for thrombin purification (Patel et al. Biochim.Biophys. Acta 748,321 (1983)). Human prethrombin 1 was activated with the purified activator from Echis carinatus venom in the presence of Ca++;, benzamidine and FPRal gel at pH 7.8. After exhaustive washing the MT1 was eluted with 0.1 M hydroxylamine in 0.15 M Na acetate buffer, pH 5.5. Under these conditions the MT1 is stable and can bestored at -70°C. Upon changing the pH of the preparation to 8.0, complete conversion into aTH occurred atroom temperature within 48 hours. Homogeneity of both preparations wasdemonstrated by PAGE. The Km and ke, values for MT1 measured on Tos-Gly-Pro-Arg pNA(0.1 M NaCl, 0.01 M TRIS, 0.01 M HEPES, 0.1% PEG, pH 7.8, 25°C) were 15.7 /iM and 126 s-1. The kinetic con stants for the aTH resulting from autocatalytic degradation of MT1 were indistinguishable from those previously established forαTH obtained by Xa activation i.e. 4.77 /μM and 126 s-1. Clotting activity of MT1 was found to be only one fifth as high as that of the resulting μTH(746 u/mg vs. 3900 u/mg as tested using the NIH standard) .Inhibitionof MTl by antithrombin III was alsomuch less rapid than αTH andmost importantly, it was not affected by high affinity heparin( Mr20,300). Under conditions of the experiment (0.3 M NaCl, 0.0rl M TRIS, 0.01 M HEPES, 2.5 mM EDTA, 0.1% PEG, pH 7.8, 25°C; [ATIII] 100 nM, [E] 10 nM), the pseudo first order rate constants in the absence of heparin were 4.04 × 10-3V1 (MTl) and 1.13 × 10-3V1 (αTH), giving apparent second order rate constants of 4.04 × 103 and 1.13 × 10-4M-1s-1. In the presence of 4.5 nM of heparin the observed first order rate constant for MTl remained unchanged whereas it increased to 6.241 × 10-3s-1 (5.5 fold) for αTH. This apparent lack of an effect of heparin may be of significance in vivo.Supported by a Matching Grant from the American National Red Cross and by the Southeastern Michigan Blood Service.

Kinetics of reconstitution of apotyrosinase by copper

1990 ◽  
Vol 68 (2) ◽  
pp. 476-479
Author(s):  
Donald C. Wigfield ◽  
Douglas M. Goltz
Keyword(s):  
Rate Constant ◽  
Copper Concentration ◽  
Copper Ions ◽  
Copper Ion ◽  
Order Rate Constant ◽  
First Order ◽  
Order Rate ◽  
Pseudo First Order ◽  
Rate Limiting ◽  
Kinetics Of

The kinetics of the reconstitution reaction of apotyrosinase with copper (II) ions are reported. The reaction is pseudo first order with respect to apoenzyme and the values of these pseudo first order rate constants are reported as a function of copper (II) concentration. Two copper ions bind to apoenzyme, and if the second one is rate limiting, the kinetically relevant copper concentration is the copper originally added minus the amount used in binding the first copper ion to enzyme. This modified copper concentration is linearly related to the magnitude of the pseudo first order rate constant, up to a copper concentration of 1.25 × 10−4 M (10-fold excess), giving a second order rate constant of 7.67 × 102 ± 0.93 × 102 M−1∙s−1.Key words: apotyrosinase, copper, tyrosinase.

scholarly journals Inactivation of the endogenous argininosuccinate lyase activity of duck δ-crystallin by modification of an essential histidine residue with diethyl pyrocarbonate

1993 ◽  
Vol 293 (2) ◽  
pp. 537-544 ◽  
Author(s):  
H J Lee ◽  
S H Chiou ◽  
G G Chang
Keyword(s):  
Enzyme Activity ◽  
Rate Constant ◽  
Order Rate Constant ◽  
Histidine Residue ◽  
Argininosuccinate Lyase ◽  
First Order ◽  
Diethyl Pyrocarbonate ◽  
Order Rate ◽  
Lyase Activity ◽  
Pseudo First Order

The argininosuccinate lyase activity of duck delta-crystallin was inactivated by diethyl pyrocarbonate at 0 degrees C and pH 7.5. The inactivation followed pseudo-first-order kinetics after appropriate correction for the decomposition of the reagent during the modification period. The plot of the observed pseudo-first-order rate constant versus diethyl pyrocarbonate concentration in the range of 0.17-1.7 mM was linear and went through the origin with a second-order rate constant of 1.45 +/- 0.1 M-1.s-1. The double-logarithmic plot was also linear, with slope of 1.13, which suggested a 1:1 stoichiometry for the reaction between diethyl pyrocarbonate and delta-crystallin. L-Arginine, L-norvaline or L-citrulline protected the argininosuccinate lyase activity of delta-crystallin from diethyl pyrocarbonate inactivation. The dissociation constants for the delta-crystallin-L-arginine and delta-crystallin-L-citrulline binary complexes, determined by the protection experiments, were 4.2 +/- 0.2 and 0.12 +/- 0.04 mM respectively. Fumarate alone had no protective effect. However, fumarate plus L-arginine gave synergistic protection with a ligand binding interacting factor of 0.12 +/- 0.02. The double-protection data conformed to a random Uni Bi kinetic mechanism. Fluorescence-quenching studies indicated that the modified delta-crystallin had minimum, if any, conformational changes as compared with the native delta-crystallin. Inactivation of the enzyme activity was accompanied by an increasing absorbance at 240 nm of the protein. The absorption near 280 nm did not change. Treatment of the modified protein with hydroxylamine regenerated the enzyme activity to the original level. These results strongly indicated the modification of an essential histidine residue. Calculation from the 240 nm absorption changes indicated that only one histidine residue per subunit was modified by the reagent. This super-active histidine residue has a pKa value of approximately 6.8 and acts as a general acid-base catalyst in the enzyme reaction mechanism. Our experimental data are compatible with an E1cB mechanism [Raushel (1984) Arch. Biochem. Biophys. 232, 520-525] for the argininosuccinate lyase with the essential histidine residue close to the arginine-binding domain of delta-crystallin. L-Citrulline, after binding to this domain, might form an extra hydrogen bond with the essential histidine residue.

Sign in / Sign up

Export Citation Format

Share Document