scholarly journals Kinetic properties of spermine synthase from bovine brain

1983 ◽  
Vol 215 (3) ◽  
pp. 669-676 ◽  
Author(s):  
R L Pajula
Keyword(s):  
Initial Velocity ◽  
Substrate Inhibition ◽  
Product Inhibition ◽  
Bovine Brain ◽  
Kinetic Properties ◽  
Methylthioadenosine Phosphorylase ◽  
Spermine Synthase ◽  
Michaelis Constants ◽  
Inhibition Studies ◽  
Competitive Product

A kinetic analysis including initial-velocity and product-inhibition studies were performed with spermine synthase purified from bovine brain. The enzyme activity was assayed in the presence of 5′-methylthioadenosine phosphorylase as an auxiliary enzyme to prevent the accumulation of the inhibitory product, 5′-methylthioadenosine, and thus to obtain linearity of the reaction with time. Initial-velocity studies gave intersecting or converging linear double-reciprocal plots. No substrate inhibition by decarboxylated S-adenosylmethionine was observed at concentrations up to 0.4 mM. Apparent Michaelis constants were 60 microM for spermidine and 0.1 microM for decarboxylated S-adenosylmethionine. Spermine was a competitive product inhibitor with respect to decarboxylated S-adenosylmethionine, but a mixed one with respect to the other substrate, spermidine. 5′-Methylthioadenosine showed a mixed inhibition with both substrates, predominantly competitive with respect to decarboxylated S-adenosylmethionine and predominantly uncompetitive with respect to spermidine. The observed kinetic and inhibition patterns are consistent with a compulsory-order mechanism, where both substrates add to the enzyme before products can be released.

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 kinetics of malonyl-CoA synthetase from Bradyrhizobium japonicum and evidence for malonyl-AMP formation in the reaction

1994 ◽  
Vol 297 (2) ◽  
pp. 327-333 ◽  
Author(s):  
Y S Kim ◽  
S W Kang
Keyword(s):  
Steady State ◽  
Bradyrhizobium Japonicum ◽  
Initial Velocity ◽  
Catalytic Mechanism ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Steady State Kinetics ◽  
Malonyl Coa ◽  
Michaelis Constants ◽  
Inhibition Studies

Malonyl-CoA synthetase catalyses the formation of malonyl-CoA directly from malonate and CoA with hydrolysis of ATP into AMP and PP1. The catalytic mechanism of malonyl-CoA synthetase from Bradyrhizobium japonicum was investigated by steady-state kinetics. Initial-velocity studies and the product-inhibition studies with AMP and PPi strongly suggested ordered Bi Uni Uni Bi Ping Pong Ter Ter system as the most probable steady-state kinetic mechanism of malonyl-CoA synthetase. Michaelis constants were 61 microM, 260 microM and 42 microM for ATP, malonate and CoA respectively, and the value for Vmax, was 11.2 microM/min. The t.l.c. analysis of the 32P-labelled products in a reaction mixture containing [gamma-32P]ATP in the absence of CoA showed that PPi was produced after the sequential addition of ATP and malonate. Formation of malonyl-AMP, suggested as an intermediate in the kinetically deduced mechanism, was confirmed by the analysis of 31P-n.m.r. spectra of an AMP product isolated from the 18O-transfer experiment using [18O]malonate. The 31P-n.m.r. signal of the AMP product appeared at 0.024 p.p.m. apart from that of [16O4]AMP, indicating that one atom of 18O transferred from [18O]malonate to AMP through the formation of malonyl-AMP. Formation of malonyl-AMP was also confirmed through the t.l.c. analysis of reaction mixture containing [alpha-32P]ATP. These results strongly support the ordered Bi Uni Uni Bi Pin Pong Ter Ter mechanism deduced from initial-velocity and product-inhibition studies.

scholarly journals Purification and catalytic properties of l-valine dehydrogenase from Streptomyces cinnamonensis

1989 ◽  
Vol 261 (3) ◽  
pp. 853-861 ◽  
Author(s):  
N D Priestley ◽  
J A Robinson
Keyword(s):  
Initial Velocity ◽  
Reductive Amination ◽  
Catalytic Properties ◽  
Product Inhibition ◽  
Aminobutyric Acid ◽  
Oxidative Deamination ◽  
Streptomyces Cinnamonensis ◽  
Michaelis Constants ◽  
Pyrimidine Bases ◽  
Inhibition Studies

NAD+-dependent L-valine dehydrogenase was purified 180-fold from Streptomyces cinnamonensis, and to homogeneity, as judged by gel electrophoresis. The enzyme has an Mr of 88,000, and appears to be composed of subunits of Mr 41,200. The enzyme catalyses the oxidative deamination of L-valine, L-leucine, L-2-aminobutyric acid, L-norvaline and L-isoleucine, as well as the reductive amination of their 2-oxo analogues. The enzyme requires NAD+ as the only cofactor, which cannot be replaced by NADP+. The enzyme activity is significantly decreased by thiol-reactive reagents, although purine and pyrimidine bases, and nucleotides, do not affect activity. Initial-velocity and product-inhibition studies show that the reductive amination proceeds through a sequential ordered ternary-binary mechanism; NADH binds to the enzyme first, followed by 2-oxoisovalerate and NH3, and valine is released first, followed by NAD+. The Michaelis constants are as follows; L-valine, 1.3 mM; NAD+, 0.18 mM; NADH, 74 microM; 2-oxoisovalerate, 0.81 mM; and NH3, 55 mM. The pro-S hydrogen at C-4′ of NADH is transferred to the substrate; the enzyme is B-stereospecific. It is proposed that the enzyme catalyses the first step of valine catabolism in this organism.

scholarly journals Kinetic analysis of duck ε-crystallin, a lens structural protein with lactate dehydrogenase activity

1990 ◽  
Vol 267 (1) ◽  
pp. 51-58 ◽  
Author(s):  
S H Chiou ◽  
H J Lee ◽  
G G Chang
Keyword(s):  
Lactate Dehydrogenase ◽  
Kinetic Analysis ◽  
Substrate Inhibition ◽  
Product Inhibition ◽  
Enzyme Mechanism ◽  
Kinetic Properties ◽  
Structural Protein ◽  
Lactate Dehydrogenase Activity ◽  
Chicken Heart ◽  
Inhibition Studies

Biochemical characterization and kinetic analysis of epsilon-crystallin from the lenses of common ducks were undertaken to elucidate the enzyme mechanism of this unique crystallin with lactate dehydrogenase (LDH) activity. Despite the structural similarities between epsilon-crystallin and chicken heart LDH, differences in charge and kinetic properties were revealed by isoenzyme electrophoresis and kinetic studies. Bi-substrate kinetic analysis examined by initial-velocity and product-inhibition studies suggested a compulsory ordered Bi Bi sequential mechanism with NADH as the leading substrate followed by pyruvate. The products were released in the order L-lactate and NAD+. The catalysed reaction is shown to have a higher rate in the formation of L-lactate and NAD+. Substrate inhibition was observed at high concentrations of pyruvate and L-lactate for the forward and reverse reactions respectively. The substrate inhibition was presumably due to the formation of epsilon-crystallin-NAD(+)-pyruvate or epsilon-crystallin-NADH-L-lactate abortive ternary complexes, as suggested by the product-inhibition studies. The significance and the interrelationship of duck epsilon-crystallin with other well-known LDHs are discussed with special regard to its role as a structural protein with some enzymic function in lens metabolism.

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 The kinetic properties and reaction mechanism of histamine methyltransferase from human skin

1980 ◽  
Vol 187 (3) ◽  
pp. 819-828 ◽  
Author(s):  
D M Francis ◽  
M F Thompson ◽  
M W Greaves
Keyword(s):  
Reaction Mechanism ◽  
Human Skin ◽  
Substrate Inhibition ◽  
Kinetic Properties ◽  
Sequential Reaction ◽  
Methyl Group ◽  
Michaelis Constants ◽  
Histamine Methyltransferase ◽  
The Mean ◽  
Inhibited Enzyme

The substrate kinetic properties of histamine methyltransferase from human skin were studied at limiting concentrations of both histamine and S-adenosylmethionine. Substrate inhibition by histamine was observed at concentrations above 10 microM. Primary plots showed evidence of a sequential reaction mechanism. The Michaelis constants were derived from secondary plots of slopes from the primary plots ([S]/v versus [S]) versus reciprocal of the second substrate concentration. The mean Km values for histamine and S-adenosylmethionine were 4.2 and 1.8 microM respectively. Histamine in concentrations of 25-100 microM inhibited enzyme activity uncompetitively with respect to S-adenosylmethionine. No substrate inhibition was observed with S-adenosylmethionine. To elucidate the reaction mechanism further, inhibition by the two products, S-adenosylhomocysteine and 1-methylhistamine, was studied. S-Adenosylhomocysteine inhibited non-competitively with respect to histamine and competitively with respect to S-adenosylmethionine. 1-Methylhistamine inhibited non-competitively with respect to histamine and to S-adenosylmethionine. These results are interpreted as providing evidence for an ordered sequential Bi Bi reaction mechanism, with the methyl-group donor S-adenosylmethionine as the first substrate that adds to the enzyme and histamine as the second substrate. 1-Methylhistamine is the first product to leave the enzyme and S-adenosylhomocysteine is the second. The results are discussed in terms of the possible role that this enzyme could play in the modulation of histamine-mediated reactions in skin.

scholarly journals The mechanism of the reaction between glutathione and 1-menaphthyl sulphate catalysed by a glutathione S-transferase from rat liver

1973 ◽  
Vol 135 (4) ◽  
pp. 797-804 ◽  
Author(s):  
Brian Gillham
Keyword(s):  
Rat Liver ◽  
Gel Filtration ◽  
Product Inhibition ◽  
Glutathione S Transferase ◽  
Michaelis Constant ◽  
Dead End ◽  
Michaelis Constants ◽  
Inhibition Studies ◽  
Inhibition Pattern ◽  
Mechanism Of The Reaction

1. The glutathione S-transferase that catalyses the reaction of 1-menaphthyl (naphth-1-ylmethyl) sulphate with GSH was purified 76-fold from rat liver. 2. The properties of the purified enzyme were studied by gel filtration and isoelectric focusing. 3. The initial-velocity pattern in the absence of products and the product-inhibition pattern have been determined. These are consistent with an Ordered Bi Bi mechanism in which the GSH adds to the enzyme before 1-menaphthyl sulphate and the products are released in the order SO42−followed by S-(1-menaphthyl)glutathione. 4. Dead-end-inhibition studies with p-aminobenzoic acid, which has been shown to be competitive with GSH and non-competitive with 1-menaphthyl sulphate, support the suggestion that an Ordered Bi Bi mechanism is operative. 5. Values were determined for some of the dissociation and Michaelis constants for the reaction of the substrates and products with the enzyme. 6. It appears that S-(1-menaphthyl)glutathione activates the enzyme when the concentration of GSH is saturating and that of 1-menaphthyl sulphate is low (of the order of its Michaelis constant).

scholarly journals A steady-state kinetic study of the reaction catalysed by the secondary-amine mono-oxygenase of Pseudomonas aminovorans

1976 ◽  
Vol 157 (1) ◽  
pp. 197-205 ◽  
Author(s):  
D F Brook ◽  
P J Large
Keyword(s):  
Steady State ◽  
Affinity Chromatography ◽  
Secondary Amine ◽  
Gel Filtration ◽  
Product Inhibition ◽  
Steady State Kinetic ◽  
Ping Pong ◽  
Michaelis Constants ◽  
Inhibition Studies ◽  
State Kinetic

1. Secondary-amine mono-oxygenase (proposed EC group 1.14.99.-) was partially purified from trimethylamine-grown Pseudomonas aminovorans by (NH4)2SO4 fractionation, gel filtration, hydrophobic chromatography on 5-aminopentylamino-Sepharose, and affinity chromatography on Sepharose-bound NADH. 2. Some problems in the affinity-chromatography step are discussed. 3. A steady-state kinetic analysis varying substrate, oxygen and electron-donor concentrations was performed, which, over the concentration range studied, gave a series of families of approximately parallel double-reciprocal plots. From secondary and tertiary plots, Michaelis constants of 0.160 mM, 0.086 mM and 0.121 mM were obtained for dimethylamine, NADPH and oxygen respectively. 4. Product-inhibition studies supported the postulated Hexa Uni Ping Pong (triple-transfer) reaction mechanism.

Glutamate Dehydrogenase from Medicago sativa L., Purification and Comparative Kinetic Studies of the Organ-Specific Multiple Forms

1980 ◽  
Vol 35 (5-6) ◽  
pp. 406-415 ◽  
Author(s):  
Marianne Nagel ◽  
Hartmann
Keyword(s):  
Medicago Sativa ◽  
Glutamate Dehydrogenase ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Kinetic Properties ◽  
Enzyme Preparations ◽  
Organ Specific ◽  
Nad Oxidoreductase ◽  
Medicago Sativa L ◽  
Inhibition Studies

Abstract NAD-specific glutamate dehydrogenase [L-glutamate: NAD + oxidoreductase (deaminating) EC 1.4.1.2] from Medicago sativa constitutes organ-specific patterns of isoenzymes. The isoenzyme-pattems of seeds (GDH-I) and roots (GDH-II) were purified 1520-fold and 92-fold, respectively. All isoenzymes of both patterns remain stable throughout the purification procedures. Isoenzyme a7, the only isoenzyme common to both patterns was isolated from the GDH-I pattern. The three enzyme preparations were found to be identical in pH optima, substrate specificity and general kinetic properties. A comparative kinetic analysis revealed no pronounced differences between the various kinetic constants evaluated for the three enzyme preparations. Furthermore an identical order of substrate binding and product release could be established. Both initial rate measure­ ments and product inhibition studies are consistent with an ordered ternary-binary kinetic mecha­ nism. The results suggest that tissue-specific enzyme multiplicity of plant glutamate dehydrogenase is not related to differences in general or kinetic properties.

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