scholarly journals Kinetic mechanism of octopus hepatopancreatic glutathione transferase in reverse micelles

1996 ◽  
Vol 315 (2) ◽  
pp. 599-606 ◽  
Author(s):  
Shiao-Shek TANG ◽  
Gu-Gang CHANG
Keyword(s):  
Aqueous Solution ◽  
Steady State ◽  
Amino Acid ◽  
Reverse Micelles ◽  
Glutathione Transferase ◽  
Substrate Inhibition ◽  
Enzyme Reaction ◽  
Kinetic Properties ◽  
Pka Value

Octopus glutathione transferase (GST) was enzymically active in aerosol-OT [sodium bis-(2-ethylhexyl)sulphosuccinate]/iso-octane reverse micelles albeit with lowered catalytic constant (kcat). The enzyme reaction rate was found to be dependent on the [H2O]/[surfactant] ratio (ωo) of the system with maximum rate observed at ωo 13.88, which corresponded to vesicles with a core volume of 64 nm3. According to the physical examinations, a vesicle of this size is barely large enough to accommodate a monomeric enzyme subunit. Dissociation of the enzyme in reverse micelles was confirmed by cross-linking of the associated subunits with glutaraldehyde and separation of the monomers and dimers with electrophoresis in the presence of SDS. The kinetic properties of the enzyme were investigated by steady-state kinetic analysis. Both GSH and 1-chloro-2,4-dinitrobenzene (CDNB) showed substrate inhibition and the Michaelis constant for CDNB was increased by 36-fold to 11.05 mM in reverse micelles. Results on the initial-velocity and product-inhibition studies indicate that the octopus GST conforms to a steady-state sequential random Bi Bi mechanism. The results from a log kcat versus pH plot suggest that amino acid residues with pKa values of 6.56±0.07 and 8.81±0.17 should be deprotonated to give optimum catalytic function. In contrast, the amino acid residue with a pKa value of 9.69±0.16 in aqueous solution had to be protonated for the reaction to proceed. We propose that the pKa1 (6.56) is that for the enzyme-bound GSH, which has a pKa value lowered by 1.40–1.54 pH units compared with that of free GSH in reverse micelles. The most probable candidate for the observed pKa2 (8.81) is Tyr7 of GST. The pKa of Tyr7 is 0.88 pH unit lower than that in aqueous solution and is about 2 pH units below the normal tyrosine. This tyrosyl residue may act as a base catalyst facilitating the dissociation of enzyme-bound GSH. The possible interaction of GST with plasma membrane in vivo is discussed.

GABA receptors precede glutamate receptors in hypothalamic development; differential regulation by astrocytes

1995 ◽  
Vol 74 (4) ◽  
pp. 1473-1484 ◽  
Author(s):  
G. Chen ◽  
P. Q. Trombley ◽  
A. N. van den Pol
Keyword(s):  
Steady State ◽  
Amino Acid ◽  
Glutamate Receptors ◽  
Gaba Receptors ◽  
Receptor Expression ◽  
Glycine Receptors ◽  
Hypothalamic Neurons ◽  
Amino Acid Receptors

1. The developmental changes in gamma-aminobutyrate (GABA)-, glutamate-, and glycine-mediated currents in cultured embryonic neurons (n = 134) from rat hypothalamus were studied with the use of whole cell voltage-clamp recording. 2. GABA-evoked currents were detected in neurons cultured from 15-day embryos (E15) a few hours after plating. Every neuron studied from the time of plating at E15 to 2 wk later responded to GABA (30 microM). The peak and steady-state currents evoked by GABA increased by four- to fivefold within 2 wk in culture. The time constants of the desensitization of GABA currents did not change during this period. The properties of the responses to GABA were not altered by different culture densities or substrates. 3. Glycine activated receptors that were pharmacologically distinct from GABA receptors on hypothalamic neurons. The glycine responses increased by > 50-fold within 2 wk in culture. The percentage of cells responding to glycine (500 microM) was 20% at 0 days in vitro (DIV), and increased to 100% at 6 DIV. Astrocytes increased both the amplitude of glycine-mediated currents and the percentage of cells responding to glycine. 4. Glutamate-mediated currents developed later than GABA-mediated currents. The percentage of cells responding to glutamate (500 microM) increased within the 1st wk, from 20% on the day of plating to 100% after 6 DIV. Both the peak currents and the steady-state currents mediated by glutamate increased by 20-fold during the 2 wk in culture. Both the amplitude of the responses to glutamate and the percentage of cells responding to glutamate were increased by growing neurons either on an astrocyte substrate or in high-density cultures. 5. The currents and conductance changes elicited by GABA were greater than those generated by glutamate or glycine throughout the period examined. This difference was particularly evident in younger cells. After 3 days in vitro, GABA (30 microM) elicited a mean current of 1,648 pA, whereas glutamate (500 microM) only elicited a 266-pA current, and glycine (500 microM) elicited a 278-pA current from neurons growing on an astrocyte layer. 6. The expression of amino acid receptors was heterogeneous among hypothalamic neurons in younger cultures. Whereas all neurons expressed GABA receptors, some developing neurons did not express detectable glutamate receptors or glycine receptors. 7. Each of the three amino acid-evoked currents increased from E15 (1 DIV) to E20 (1 DIV), indicating an intrinsic development in the expression of the amino acid receptors in vivo. The GABA, glutamate, and glycine currents at E15, 10 DIV were similar to the currents at E20, 5 DIV (both 25 days after conception), suggesting parallel developmental patterns for amino acid receptor expression in vitro and in vivo. 8. Together, these data suggest that GABA may play a major role in early development because hypothalamic neurons are more sensitive to GABA than to either glutamate or glycine. However, glutamate and glycine receptors appear more sensitive to regulation by the local environment than GABA receptors because culture density and the astrocyte substrate have greater inductive effects on glutamate and glycine receptors than on GABA receptors.

scholarly journals Purification and kinetic properties of ox brain histamine N-methyltransferase

1986 ◽  
Vol 233 (3) ◽  
pp. 669-676 ◽  
Author(s):  
W L Gitomer ◽  
K F Tipton
Keyword(s):  
Acetic Acid ◽  
Steady State ◽  
Mouse Brain ◽  
Initial Rate ◽  
Substrate Inhibition ◽  
Gel Filtration ◽  
Product Inhibition ◽  
Kinetic Properties ◽  
Brain Histamine ◽  
Inhibition Studies

Histamine N-methyltransferase (EC 2.1.1.8) was purified 1100-fold from ox brain. The native enzyme has an Mr of 34800 +/- 2400 as measured by gel filtration on Sephadex G-100. The enzyme is highly specific for histamine. It does not methylate noradrenaline, adrenaline, DL-3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, 3-hydroxytyramine or imidazole-4-acetic acid. Unlike the enzyme from rat and mouse brain, ox brain histamine N-methyltransferase did not exhibit substrate inhibition by histamine. Initial rate and product inhibition studies were consistent with an ordered steady-state mechanism with S-adenosylmethionine being the first substrate to bind to the enzyme and N-methylhistamine being the first product to dissociate.

scholarly journals Steady-state and pre-steady kinetic studies on mitochondrial sheep liver aldehyde dehydrogenase. A comparison with the cytoplasmic enzyme

1978 ◽  
Vol 171 (3) ◽  
pp. 527-531 ◽  
Author(s):  
A K H MacGibbon ◽  
L F Blackwell ◽  
P D Buckley
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Kinetic Studies ◽  
Kinetic Properties ◽  
Protein Fluorescence ◽  
Sheep Liver ◽  
Steady State Kinetic ◽  
Wide Range ◽  
Liver Aldehyde Dehydrogenase

Kinetic studies were carried out on mitochondrial aldehyde dehydrogenase (EC 1.2.1.3) isolated from sheep liver. Steady-state studies over a wide range of acetaldehyde concentrations gave a non-linear double-reciprocal plot. The dissociation of NADH from the enzyme was a biphasic process with decay constants 0.6s-1 and 0.09s-1. Pre-steady-state kinetic data with propionaldehyde as substrate could be fitted by using the same burst rate constant (12 +/- 3s-1) over a wide range of propionaldehyde concentrations. The quenching of protein fluorescence on the binding of NAD+ to the enzyme was used to estimate apparent rate constants for binding (2 × 10(4) litre.mol-1.s-1) and dissociation (4s-1). The kinetic properties of the mitochondrial enzyme, compared with those reported for the cytoplasmic aldehyde dehydrogenase from sheep liver, show significant differences, which may be important in the oxidation of aldehydes in vivo.

S-Adenosylhomocysteine Metabolism in Various Species

1975 ◽  
Vol 53 (3) ◽  
pp. 312-319 ◽  
Author(s):  
R. D. Walker ◽  
J. A. Duerre
Keyword(s):  
Rat Liver ◽  
Condensation Reaction ◽  
Substrate Inhibition ◽  
Product Inhibition ◽  
Kinetic Properties ◽  
Adenosylhomocysteine Hydrolase ◽  
In Vivo Experiments ◽  
Cleavage Enzyme

Eleven microorganisms, four plants, and major organs from the chicken, dog, rat, and rabbit were assayed for the presence of S-adenosylhomocysteine hydrolase, S-adenosyl-homocysteine nucleosidase, and S-ribosylhomocysteine-cleavage enzyme. All bacteria (procaryotes) were found to possess S-adenosylhomocysteine nucleosidase and S-ribosylhomocysteine-cleavage enzyme but not S-adenosylhomocysteine hydrolase. All eucaryotes tested, including yeasts, plants, birds, and mammals, possessed S-adenosylhomocysteine hydrolase but not S-adenosylhomocysteine nucleosidase or S-ribosylhomocysteine-cleavage enzyme. Of all the organs assayed in the vertebrates, the level of S-adenosylhomocysteine hydrolase was highest in liver, pancreas, and kidney, lower in spleen and testis, and very low in brain and heart. In all systems tested, equilibrium of the hydrolase reaction always favored synthesis over hydrolysis. We studied some of the kinetic properties of the hydrolase from rat liver. In the direction of synthesis, the Km value was 1.5 mM for adenosine and 4.5 mM for L-homocysteine, whereas marked substrate inhibition was observed with L-homocysteine. The condensation reaction is subject to product inhibition, and was inhibited by adenine. Results from in-vivo experiments revealed that the cells of the various organs of the dog are impermeable to the exogenously administered S-adenosylhomocysteine.

scholarly journals Steady-state kinetics and chemical mechanism of octopus hepatopancreatic glutathione transferase

1995 ◽  
Vol 309 (1) ◽  
pp. 347-353 ◽  
Author(s):  
S S Tang ◽  
G G Chang
Keyword(s):  
Steady State ◽  
Glutathione Transferase ◽  
Isotope Effects ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Chemical Mechanism ◽  
Octopus Vulgaris ◽  
Pka Value ◽  
Solvent Isotope ◽  
Solvent Isotope Effects

The kinetic mechanism of glutathione S-transferase (GST) from Octopus vulgaris hepatopancreas was investigated by steady-state analysis. Initial-velocity studies showed an intersecting pattern, which suggests a sequential kinetic mechanism for the enzyme. Product-inhibition patterns by chloride and the conjugate product were all non-competitive with respect to glutathione or 1-chloro-2,4-dinitrobenzene (CDNB), which indicates that the octopus digestive gland GST conforms to a steady-state sequential random Bi Bi kinetic mechanism. Dead-end inhibition patterns indicate that ethacrynic acid ([2,3-dichloro-4-(2-methyl-enebutyryl) phenoxy]acetic acid) binds at the hydrophobic H-site, norophthalmic acid (gamma-glutamylalanylglycine) binds at the glutathione G-site, and glutathione-ethacrynate conjugate occupied both H- and G-sites of the enzyme. The chemical mechanism of the enzyme was examined by pH and kinetic solvent-isotope effects. At pH (and p2H) = 8.011, in which kcat. was independent of pH or p2H, the solvent isotope effects on V and V/KmGSH were near unity, in the range 1.069-1.175. An inverse isotope effect was observed for V/KmCDNB (0.597), presumably resulting from the hydrogen-bonding of enzyme-bound glutathione, which has pKa of 6.83 +/- 0.04, a value lower by 2.34 pH units than the pKa of glutathione in aqueous solution. This lowering of the pKa value for the sulphydryl group of the bound glutathione was presumably due to interaction with the active site Tyr7, which had a pKa value of 8.46 +/- 0.09 that was raised to 9.63 +/- 0.08 in the presence of glutathione thiolate. Subsequent chemical reaction involves attacking of thiolate anion at the electrophilic substrate with the formation of a negatively charged Meisenheimer complex, which is the rate-limiting step of the reaction.

scholarly journals Purification and properties of acetyl-CoA:l-glutamate N-acetyltransferase from human liver

1982 ◽  
Vol 205 (1) ◽  
pp. 123-127 ◽  
Author(s):  
C Bachmann ◽  
S Krähenbühl ◽  
J P Colombo
Keyword(s):  
Amino Acid ◽  
Human Liver ◽  
Deae Cellulose ◽  
Forward Direction ◽  
Liver Mitochondria ◽  
Kinetic Properties ◽  
Urea Synthesis ◽  
Acetyl Coa

Acetyl-CoA:L-glutamate N-acetyltransferase (amino acid acetyltransferase, EC 2.3.1.1) was isolated from human liver mitochondria by precipitation with (NH4)2SO4 and chromatography on hydroxyapatite, DEAE-cellulose and Sephacryl 300. This gave a 360-fold purification. The molecular weight was estimated to be approx. 190 000. The kinetic properties in the absence of arginine are compatible with a rapid-equilibrium random Bi Bi mechanism. The estimated constants are: for the substrates Km, acetyl-CoA 4.4 mM, Ki, acetyl-CoA 4.7 mM, Km, glutamate 8.1 mM, Ki, glutamate 8.8 mM; for the products, Ki, acetylglutamate 0.28 mM, Ki, CoA 5.6 mM. The rate constant for the forward direction is 1.24s-1. If in vivo the constants are of the same order of magnitude as in vitro, the synthesis of N-acetylglutamate, an obligate activator of the first step of urea synthesis, can be expected to occur in the mitochondrion under conditions where the amino acid acetyltransferase is not saturated by its substrates. The regulation of the first step of urea synthesis could thus depend mainly on the intramitochondrial substrate and perhaps product concentrations of amino acid acetyltransferase.

Amino acid exchange between plasma and erythrocytes in vivo in humans

1989 ◽  
Vol 67 (6) ◽  
pp. 2383-2388 ◽  
Author(s):  
D. Darmaun ◽  
P. Froguel ◽  
M. Rongier ◽  
J. J. Robert
Keyword(s):  
Steady State ◽  
Amino Acid ◽  
Whole Blood ◽  
Gas Chromatography Mass Spectrometry ◽  
Confidence Limits ◽  
Amino Acid Exchange ◽  
High Concentration ◽  
Amino Acid Levels ◽  
Acid Exchange

To study amino acid exchange between plasma and erythrocytes in vivo, 4-h primed, continuous intravenous infusions of L-[1-13C]leucine, [15N]glycine, and L-[15N]alanine were administered to five healthy young men in the postabsorptive state. Stable isotope enrichments and amino acid levels were determined by gas chromatography-mass spectrometry in both plasma and whole blood and estimated (using hematocrit) in erythrocytes. A high concentration gradient across the erythrocyte membrane was consistently found for glycine (552 +/- 268 microM in erythrocytes vs. 155 +/- 35 microM in plasma), but not for leucine or alanine. A steady-state isotopic enrichment was observed in whole blood as well as plasma for each amino acid in every subject. Steady-state [13C]leucine enrichment in erythrocytes did not differ from plasma enrichment at steady state, the ratio of erythrocyte to plasma enrichment being 1.03 +/- 0.20 (95% confidence limits = 0.78-1.28); in contrast, this ratio reached only 0.23 +/- 0.04 and 0.59 +/- 0.09 (confidence limits 0.18-0.28 and 0.48-0.70) for [15N]glycine and [15N]alanine at steady state, respectively. These results suggest that most of erythrocyte leucine is exchangeable with plasma, whereas only a fraction of erythrocyte glycine and alanine is involved in exchange with plasma in vivo.

scholarly journals A general method to predict the effect of single amino acid substitutions on enzyme catalytic activity

2017 ◽  
Author(s):  
Yu-Hsiu T. Lin ◽  
Cheng Lai Victor Huang ◽  
Christina Ho ◽  
Max Shatsky ◽  
Jack F. Kirsch
Keyword(s):  
Amino Acid ◽  
Kinetic Parameters ◽  
Active Site ◽  
Enzyme Reaction ◽  
Site Directed Mutagenesis ◽  
Enzyme Function ◽  
Single Amino Acid ◽  
Kinetic Properties ◽  
Amino Acid Substitutions ◽  
Active Site Residues

ABSTRACTOver the past thirty years, site-directed mutagenesis has become established as one of the most powerful techniques to probe enzyme reaction mechanisms1-3. Substitutions of active site residues are most likely to yield significant perturbations in kinetic parameters, but there are many examples of profound changes in these values elicited by remote mutations4-6. Ortholog comparisons of extant sequences show that many mutations do not have profound influence on enzyme function. As the number of potential single natural amino acid substitutions that can be introduced in a protein of N amino acids in length by directed mutation is very large (19 * N), it would be useful to have a method to predict which amino acid substitutions are more likely to introduce significant changes in kinetic parameters in order to design meaningful probes into enzyme function. What is especially desirable is the identification of critical residues that do not contact the substrate directly, and may be remote from the active site.We collected literature data reflecting the effects of 2,804 mutations on kinetic properties for 12 enzymes. These data along with characteristic predictors were used in a machine-learning scheme to train a classifier to predict the effect of mutation. Use of this algorithm allows one to predict with a 2.5-fold increase in precision, if a given mutation, made anywhere in the enzyme, will cause a decrease in kcat/Km value of ≥ 95%. The improved precision allows the experimentalist to reduce the number of mutations necessary to probe the enzyme reaction mechanism.

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