scholarly journals Studies of the specificity and kinetics of rat liver spermidine/spermine N1-acetyltransferase

1983 ◽  
Vol 213 (3) ◽  
pp. 701-706 ◽  
Author(s):  
F Della Ragione ◽  
A E Pegg
Keyword(s):  
Rat Liver ◽  
Kinetic Mechanism ◽  
Competitive Inhibitor ◽  
Aliphatic Chain ◽  
Amino Group ◽  
Acetyl Coa ◽  
Competitive Kinetics ◽  
Primary Amino ◽  
Secondary Amino ◽  
Kinetics Of

The substrate specificity and kinetic mechanism of spermidine N1-acetyltransferase from rat liver was investigated using a highly purified (18 000-fold) preparation from the livers of rats in which the enzyme was induced by treatment with carbon tetrachloride (1.5 ml/kg body wt. 6h before death). The enzyme catalysed the acetylation of spermidine, spermine, sym-norspermidine, sym-norspermine, N-(3-aminopropyl)-cadaverine, N1-acetylspermine, 3,3′-diamino-N-methyldipropylamine and 1,3-diaminopropane, but was inactive with putrescine, cadaverine, sym-homospermidine and N1-acetylspermidine. These results suggest that the enzyme is highly specific for the acetylation of a primary amino group that is separated by a three-carbon aliphatic chain from another nitrogen atom (i.e. the substrates are of the type H2N[CH2]3NHR). The maximal rates of acetylation of 1,3-diaminopropane and 3,3′-diamino-N-methyldipropylamine were much lower than the maximal rates with spermidine or sym-norspermidine as substrates, suggesting a preference for a secondary amino group bearing the aminopropyl group that is acetylated. The best substrates for acetylation were sym-norspermidine and sym-norspermine, which had Km values of about 10 micrograms and Vmax. values of about 2 mumol of product/min per mg of enzyme compared with Km of 130 microM and Vmax. of 1.3 mumol/min per mg for spermidine. N1-Acetylspermidine (the product of the reaction) and N8-acetylspermidine were weak inhibitors and were competitive with spermidine, having Ki values of about 6.6 mM and 0.4 mM respectively. N1-Acetylspermidine was a non-competitive inhibitor with respect to acetyl-CoA. CoA was also inhibitory to the reaction, showing non-competitive kinetics when either [acetyl-CoA] or [spermidine] was varied. These results suggest that the reaction occurs via an ordered Bi Bi mechanism in which spermidine binds first and N1-acetyl-spermidine is the final product to be released.

scholarly journals The kinetic mechanism and properties of the cytoplasmic acetoacetyl-coenzyme A thiolase from rat liver

1974 ◽  
Vol 139 (1) ◽  
pp. 109-121 ◽  
Author(s):  
B. Middleton
Keyword(s):  
Rat Liver ◽  
Substrate Inhibition ◽  
Kinetic Mechanism ◽  
Competitive Inhibitor ◽  
Acetyl Coa ◽  
Keto Form ◽  
Ping Pong ◽  
Substrate Inhibitor ◽  
The Hill ◽  
Covalent Intermediate

1. Cytoplasmic acetoacetyl-CoA thiolase was highly purified in good yield from rat liver extracts. 2. Mg2+ inhibits the rate of acetoacetyl-CoA thiolysis but not the rate of synthesis of acetoacetyl-CoA. Measurement of the velocity of thiolysis at varying Mg2+ but fixed acetoacetyl-CoA concentrations gave evidence that the keto form of acetoacetyl-CoA is the true substrate. 3. Linear reciprocal plots of velocity of acetoacetyl-CoA synthesis against acetyl-CoA concentration in the presence or absence of desulpho-CoA (a competitive inhibitor) indicate that the kinetic mechanism is of the Ping Pong (Cleland, 1963) type involving an acetyl-enzyme covalent intermediate. In the presence of CoA the reciprocal plots are non-linear, becoming second order in acetyl-CoA (the Hill plot shows a slope of 1.7), but here this does not imply co-operative phenomena. 4. In the direction of acetoacetyl-CoA thiolysis CoA is a substrate inhibitor, competing with acetoacetyl-CoA, with a Ki of 67μm. Linear reciprocal plots of initial velocity against concentration of mixtures of acetoacetyl-CoA plus CoA confirmed the Ping Pong mechanism for acetoacetyl-CoA thiolysis. This method of investigation also enabled the determination of all the kinetic constants without complication by substrate inhibition. When saturated with substrate the rate of acetoacetyl-CoA synthesis is 0.055 times the rate of acetoacetyl-CoA thiolysis. 5. Acetoacetyl-CoA thiolase was extremely susceptible to inhibition by an excess of iodoacetamide, but this inhibition was completely abolished after preincubation of the enzyme with a molar excess of acetoacetyl-CoA. This result was in keeping with the existence of an acetyl-enzyme. Acetyl-CoA, in whose presence the overall reaction could proceed, gave poor protection, presumably because of the continuous turnover of acetyl-enzyme in this case. 6. The kinetic mechanism of cytoplasmic thiolase is discussed in terms of its proposed role in steroid biosynthesis.

scholarly journals The reaction of ornithine aminotransferase with ornithine

1982 ◽  
Vol 201 (1) ◽  
pp. 221-225 ◽  
Author(s):  
J A Williams ◽  
G Bridge ◽  
L J Fowler ◽  
R A John
Keyword(s):  
Amino Acids ◽  
Carbon Atom ◽  
Rat Liver ◽  
Dissociation Constants ◽  
Amino Group ◽  
Ornithine Aminotransferase ◽  
Mammalian Enzyme ◽  
Kinetics Of ◽  
Plant Enzyme

Rat liver ornithine aminotransferase is found to exchange the pro-S hydrogen on the delta-carbon atom of ornithine exclusively, thus showing that the mammalian enzyme transfers the delta-amino group and not the alpha-amino group as has been demonstrated with the plant enzyme [Mestichelli, Gupta & Spenser (1979) J. Biol. Chem. 254, 640-647]. The enzyme also transfers the alpha-amino group of glutamate and the kinetics of the half reactions between the enzyme and both amino acids are compared. Rate and dissociation constants for both reactions are determined.

scholarly journals Factors which affect the activity of purified rat liver acyl-CoA oxidase

1993 ◽  
Vol 290 (1) ◽  
pp. 97-102 ◽  
Author(s):  
R Hovik ◽  
H Osmundsen
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Oxidase Activity ◽  
Substrate Inhibition ◽  
Critical Micellar Concentration ◽  
Competitive Inhibitor ◽  
Acetyl Coa ◽  
Acyl Coa Oxidase ◽  
Triton X

The activity of the enzyme acyl-CoA oxidase (EC 1.3.99.3) is influenced by detergents. At concentrations above the critical micellar concentration, Triton X-100, Triton X-114 and Thesit stimulate oxidase activity. Lower concentrations of Triton X-100 and Triton X-114 render the acyl-CoA oxidase less sensitive towards substrate inhibition by palmitoyl-CoA or dec-4-cis-enoyl-CoA. Other detergents inhibited the enzyme activity. CoA was found to be a relatively powerful competitive inhibitor of the enzyme, with a Ki, slope value of 63 +/- 3 microM. This inhibition is dependent on an intact CoA molecule, as dephospho-CoA, dethio-CoA and acetyl-CoA are less potent inhibitors of the enzyme. Dec-2-trans-enoyl-CoA is a product-inhibitor of acyl-CoA oxidase, with a Ki, slope value of 7 +/- 1 microM.

scholarly journals Mechanism of the inhibition of cholesterol biosynthesis by 6-fluoromevalonate

1985 ◽  
Vol 227 (1) ◽  
pp. 247-254 ◽  
Author(s):  
J F Nave ◽  
H d'Orchymont ◽  
J B Ducep ◽  
F Piriou ◽  
M J Jung
Keyword(s):  
Rat Liver ◽  
Cholesterol Biosynthesis ◽  
Mevalonic Acid ◽  
Competitive Inhibitor ◽  
Isopentenyl Pyrophosphate ◽  
Multienzyme System ◽  
Kinetics Of

6-Fluoromevalonate blocks the incorporation of mevalonic acid, but not that of isopentenyl pyrophosphate, into non-saponifiable lipids in a rat liver multienzyme system. With 3H-labelled 6-fluoromevalonate, it was found that 6-fluoromevalonate is converted to its phospho and pyrophospho derivatives in this system. The kinetics of the two kinases were studied. 6-Fluoromevalonate 5-pyrophosphate is a potent competitive inhibitor of pyrophosphomevalonate decarboxylase (Ki 37 nM). In the multienzyme assay for cholesterol biosynthesis, there is accumulation of mevalonate 5-phosphate and mevalonate 5-pyrophosphate in the presence of 5 microM-6-fluoromevalonate, and 6-fluoromevalonate 5-pyrophosphate is more effective than 6-fluoromevalonate in inhibiting cholesterol biosynthesis. We suggest therefore that 6-fluoromevalonate blocks cholesterol biosynthesis at the level of pyrophosphomevalonate decarboxylase after being pyrophosphorylated.

scholarly journals The kinetics of phenethylhydrazine oxidation by monoamine oxidase

1971 ◽  
Vol 125 (2) ◽  
pp. 521-524 ◽  
Author(s):  
K. F. Tipton ◽  
I. P. C. Spires
Keyword(s):  
Monoamine Oxidase ◽  
Rat Liver ◽  
Competitive Inhibitor ◽  
Product Inhibition ◽  
Parallel Lines ◽  
Hydrazine Oxidation ◽  
Pig Brain ◽  
Kinetics Of

1. In the presence of the substrate benzylamine, phenethylhydrazine has been shown to be a competitive inhibitor of monoamine oxidase from rat liver and pig brain. 2. Phenethylhydrazine is also a substrate for monoamine oxidase. Reciprocal plots for hydrazine oxidation give families of intersecting lines in contrast with the parallel lines previously reported for tyramine oxidation. 3. Two possible modifications of the mechanism obeyed by tyramine oxidation are suggested, but the product inhibition results are insufficient to distinguish between these two mechanisms.

scholarly journals Some aspects of the kinetics of rat liver pyruvate carboxylase

1970 ◽  
Vol 120 (1) ◽  
pp. 79-93 ◽  
Author(s):  
Janet M. Wimhurst ◽  
K. L. Manchester
Keyword(s):  
Rat Liver ◽  
Binding Sites ◽  
Pyruvate Carboxylase ◽  
Competitive Inhibitor ◽  
Mitochondrial Fraction ◽  
Spectrophotometric Assay ◽  
Time Dependent ◽  
Malonyl Coa ◽  
Kinetics Of ◽  
Hill Plots

1. The kinetics of rat liver pyruvate carboxylase were examined and the effect of various agents as activators or inhibitors determined. 2. Essentially similar results were obtained in comparisons of kinetics determined by a radioactivity method involving extracts of acetone-dried powders from whole livers and with a spectrophotometric assay using partially purified enzyme from the mitochondrial fraction. Activity per g of liver from fed or starved rats assayed under optimum substrate and activator conditions was 3 or 6 μmol of oxaloacetate formed/min at 30°C, respectively. 3. The enzyme exhibited cold-lability and lost activity on standing, even in 1.5m-sucrose. 4. The Km towards pyruvate was about 0.33mm and towards bicarbonate 4.2mm. Km towards MgATP2− was 0.14mm. Mg2+ ions activated the enzyme, in addition to their role in MgATP2− formation. From calculations of likely concentrations of free Mg2+ in the assay medium a Ka towards Mg2+ of about 0.25mm was deduced. Mn2+ also activated the enzyme as well as Mg2+, but at much lower concentrations. It appeared to be inhibitory when concentrations of free Mn2+ as low as 0.1mm were present. 5. Excess of ATP is inhibitory, and this appears at least in part independent of the trapping of Mg2+. 6. Both Co2+ and Zn2+ were inhibitory; 2mol of the latter appeared to be bound even in the presence of excess of Mg2+ and the inhibition was time-dependent. 7. Ca2+ inhibited by competition with Mg2+ (Ki about 0.38mm). The inhibition due to Ca2+ was less pronounced when activation was with Mn2+. Inhibition by Ca2+ and ATP appeared to be additive. 8. Hill plots suggested that no interactions occurred between ATP-binding sites. Although similar plots for total Mg2+ gave n=3.6, no conclusions could be drawn due to the chelation of the cation with other components of the assay. Similar difficulties arose in assessing the values for Ca2+. 9. The enzyme was inactive in the absence of acetyl-CoA and showed a sigmoidal response in its presence. Ka was about 0.1mm with possibly up to four binding sites. Malonyl-CoA was a competitive inhibitor, with Ki 0.01mm. 10. There was no apparent inhibition by glucose, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-diphosphate, acetoacetate, β-hydroxybutyrate, malate, aspartate, pyruvate, palmitoylcarnitine, octanoate, glutathione, butacaine, triethyltin or potassium chloride under the conditions used. Inhibition was found with citrate (possibly by chelation) and adenosine, and also by phosphoenolpyruvate, AMP, ADP and cyclic AMP, Ki towards the last four being 0.55, 0.76, 0.25 and 1.4mm respectively.

scholarly journals Regulation of kinase reactions in pig heart pyruvate dehydrogenase complex

1979 ◽  
Vol 181 (2) ◽  
pp. 427-433 ◽  
Author(s):  
A L Kerbey ◽  
P M Radcliffe ◽  
P J Randle ◽  
P H Sugden
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Competitive Inhibitor ◽  
Sodium Pyruvate ◽  
Acetyl Coa ◽  
32P Incorporation ◽  
Kinetics Of ◽  
Uncompetitive Inhibitor ◽  
Dehydrogenase Complex ◽  
Concentration Ratios

1. Pig heart pyruvate dehydrogenase complex is inactivated by phosphorylation (MgATP2-) of an alpha-chain of the decarboxylase component. Three serine residues may be phosphorylated, one of which (site 1) is the major inactivating site. 2. The relative rates of phosphorylation are site 1 greater than 2 greater than site 3. 3. The kinetics of the inactivating phosphorylation were investigated by measuring inactivation of the complex with MgATP2-. The apparent Km for the Mg complex of ATP was 25.5 microM; ADP was a competitive inhibitor (Ki 69.8 microM) and sodium pyruvate an uncompetitive inhibitor (Ki 2.8 microM). Inactivation was accelerated by increasing concentration ratios of NADH/NAD+ and of acetyl-CoA/CoA. 4. The kinetics of additional phosphorylations (predominantly site 2 under these conditions) were investigated by measurement of 32P incorporation into non-radioactive pyruvate dehydrogenase phosphate containing 3-6% of active complex, and assumed from parrallel experiments with 32P labelling to contain 91% of protein-bound phosphate in site 1 and 9% in site 2. 5. The apparent Km for the Mg complex of ATP was 10.1 microM; ADP was a competitive inhibitor (Ki 31.5 microM) and sodium pyruvate an uncompetitive inhibitor (Ki 1.1 mM). 6. Incorporation was accelerated by increasing concentration ratios of NADH/NAD+ and of acetyl-CoA/CoA, although it was less marked at the highest ratios.

Acetyl-CoA hydrolase: relation between activity and cholesterol metabolism in rat

1988 ◽  
Vol 255 (5) ◽  
pp. R724-R730
Author(s):  
S. Ebisuno ◽  
F. Isohashi ◽  
Y. Nakanishi ◽  
Y. Sakamoto
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Cholesterol Metabolism ◽  
Cholesterol Biosynthesis ◽  
Physiological Role ◽  
Competitive Inhibitor ◽  
Isobutyric Acid ◽  
Enzyme Linked Immunosorbent Assay ◽  
Acetyl Coa ◽  
Coa Hydrolase

To examine whether cytosolic acetyl-CoA hydrolase in rat liver is involved in regulation of cholesterol biosynthesis, we investigated the alteration of the enzyme activity under conditions of stimulation (cholestyramine treatment) and suppression [cholesterol feeding, a potent competitive inhibitor of microsomal 3-hydroxy-3-methylglutaryl-CoA reductase (CS 514) treatment, and a hypolipidemic drug [alpha-(p-chlorophenoxy)isobutyric acid, CPIB] injection) of cholesterol biosynthesis. The enzyme activity in rat liver increased significantly in the early diabetic, cholesterol-fed, CS 514-, and CPIB-treated groups, but no change in its activity was observed in chronic diabetic groups. Cholestyramine treatment to cholesterol-fed rats made the enzyme activity return to the initial level. When chronic diabetic rats were given a cholesterol diet or treated with CS 514 or CPIB, the activity increased significantly. Inhibition of cholesterol biosynthesis caused by these treatments induced increase in the enzyme activity with increase in the enzyme protein, judging from results obtained by enzyme-linked immunosorbent assay. These results suggest that this enzyme has a physiological role in maintenance of the equilibrium between the cytosolic acetyl-CoA concentration and CoA-SH pool for cholesterol metabolism.

scholarly journals Synthesis of thiol-containing analogues of puromycin and a study of their interaction with N-acetylphenylalanyl-transfer ribonucleic acid on ribosomes to form thioesters

1975 ◽  
Vol 149 (1) ◽  
pp. 209-220 ◽  
Author(s):  
John Gooch ◽  
Arthur O. Hawtrey
Keyword(s):  
Escherichia Coli ◽  
Rat Liver ◽  
Ribonucleic Acid ◽  
Incubation Medium ◽  
Amino Group ◽  
Marked Contrast ◽  
E Coli ◽  
Primary Amino ◽  
Compound Xvii

1. The thiol-containing analogue of puromycin, 6-dimethylamino-9-{1′-[3′-(2″-mercapto-3″-phenylpropionamido)-3′-deoxy-β-d-ribofuranosyl]}purine (XVII) in which the primary amino group of the antibiotic is replaced with a thiol grouping, was synthesized chemically (compound XVII is abbreviated to thiopuromycin). 2. Thiopuromycin (XVII) was found to be active in releasing N-[3H]acetylphenylalanine from its tRNA carrier as the thioester, N-acetylphenylalanylthiopuromycin (XIX) in the Escherichia coli ribosomal system. The reaction product (XIX) was synthesized chemically from thiopuromycin and N-acetylphenylalanine and found to be stable to hydrolysis in the standard incubation medium at pH7.6. dl-Phenyl-lactylpuromycin (XXI), the hydroxy analogue of puromycin, was also synthesized chemically and shown to release N-acetylphenylalanine from its tRNA carrier in the E. coli ribosomal system, thus confirming the previous results of Fahnestock et al. [Biochemistry (1970) 9, 2477–2483]. 3. In marked contrast with the results obtained in the E. coli system, both thiopuromycin (XVII) and hydroxypuromycin (XXI) were found to be inactive in releasing N-acetylphenylalanine from its tRNA carrier in the rat liver ribosomal system.

Reaction of the anionic acetylation agent methyl acetyl phosphate with D-3-hydroxybutyrate dehydrogenase

1986 ◽  
Vol 64 (5) ◽  
pp. 434-440 ◽  
Author(s):  
Ronald Kluger ◽  
Wing-Cheong Tsui
Keyword(s):  
Ph Dependence ◽  
Competitive Inhibitor ◽  
Amino Group ◽  
Acetyl Phosphate ◽  
Hydrogen Bond Donor ◽  
Phosphate Monoesters ◽  
Dimethylmaleic Anhydride ◽  
A Site ◽  
Acyl Phosphate ◽  
Kinetics Of

Methyl acetyl phosphate is a competitive inhibitor of the reduction of acetoacetate by D-3-hydroxybutyrate dehydrogenase. The material also irreversibly inactivates the enzyme. The kinetics of the inactivation are consistent with methyl acetyl phosphate acetylating the conjugate base of a hydrogen bond donor. Protection offered by a substrate analogue (methyl acetonylphosphonate) in the presence of coenzyme implicates reaction at the cationic active site. Reversible protection by the amino group reagent 2,3-dimethylmaleic anhydride suggests that methyl acetyl phosphate reacts with an amino group. Sulfhydryl reagents and acetyl phosphate, a poorer acetylating agent, do not inactivate the enzyme. The pH dependence of the inactivation suggests that the acetylation occurs at a site that has a pKa of 8.2. The utility of methyl acetyl phosphate and other acyl phosphate monoesters in reacting with lysines adjacent to cationic sites of enzymes, hemoglobin, and histones is noted.

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