Ornithine transcarbamylase from Mycobacterium smegmatis ATCC 14468: purification, properties, and reaction mechanism

1986 ◽  
Vol 64 (12) ◽  
pp. 1349-1355 ◽  
Author(s):  
Suhail Ahmad ◽  
R. K. Bhatnagar ◽  
T. A. Venkitasubramanian
Keyword(s):  
Enzyme Activity ◽  
Reaction Mechanism ◽  
Mycobacterium Smegmatis ◽  
Kinetic Mechanism ◽  
Competitive Inhibitor ◽  
Ornithine Transcarbamylase ◽  
Carbamyl Phosphate ◽  
Michaelis Constants ◽  
Variable Substrate ◽  
Uncompetitive Inhibitor

Ornithine transcarbamylase (EC 2.1.3.3) has been purified 980-fold from Mycobacterium smegmatis and has a molecular weight of 116 000. Initial velocity determinations indicated that the reaction proceeds via a sequential kinetic mechanism. The limiting Michaelis constants for carbamyl phosphate (KmA) and ornithine (KmB) and the dissociation constant for carbamyl phospate (Kia) were found to be 0.20, 0.25, and 0.07 mM, respectively. Ornithine at higher concentrations acted as an uncompetitive inhibitor when carbamyl phosphate was the variable substrate. Phosphate was a competitive inhibitor with carbamyl phosphate as variable substrate and showed noncompetitive or mixed type inhibition when ornithine was the variable substrate. Norvaline acted as a competitive inhibitor with ornithine as variable substrate and as an uncompetitive inhibitor when carbamyl phophate was the variable substrate. Such inhibitory patterns are characteristic of reactions that proceed via sequential ordered mechanisms. Although the enzyme activity was strongly inhibited by arginine, several arginine analogs had no effect on the enzyme activity. The results suggest that, even though the enzyme from M. smegmatis is unique in the sense that it is feedback inhibited by arginine, the reaction mechanism is similar to the ornithine transcarbamylase isolated from other microorganisms.

Phosphoenolpyruvate Carboxylase of Thiobacillus thiooxidans. Kinetic and Metabolic Control Properties

1972 ◽  
Vol 50 (2) ◽  
pp. 158-165 ◽  
Author(s):  
R. L. Howden ◽  
H. Lees ◽  
Isamu Suzuki
Keyword(s):  
Enzyme Activity ◽  
Competitive Inhibitor ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Pep Carboxylase ◽  
Thiobacillus Thiooxidans ◽  
Powerful Activator ◽  
Michaelis Constants ◽  
Noncompetitive Inhibitor ◽  
Decreasing Ph

Phosphoenolpyruvate (PEP) carboxylase (orthophosphate:oxalacetate carboxy-lyase (phosphorylating), EC 4.1.1.31) was purified 19-fold from Thiobacillus thiooxidans. The level of enzyme activity was dependent on culture age. No enzyme activity could be obtained from frozen cells.The pH optimum of the enzyme was determined to be around 8.0. Apparent Michaelis constants were determined for the substrates:phosphoenolpyruvate (1.4, 1.5 mM), bicarbonate (0.4, 1.1 mM), and magnesium (1.1, 0.8 mM) at pH 7.0 and 8.0, respectively. Acetyl-CoA was found to be a powerful activator of this enzyme, with the degree of activation increasing with decreasing pH. The concentration of acetyl-CoA to obtain half-maximal activation, however, remained fairly constant and low, namely 1.2 and 1.0 μM at pH 7.0 and 8.0, respectively. L-Aspartate and L-malate were strong inhibitors of enzyme activity. In the presence of aspartate at pH 7.0 the double reciprocal activity plots for PEP became nonlinear, a characteristic of negative cooperativity. These plots became linear with the addition of acetyl-CoA with aspartate now acting as a noncompetitive inhibitor with respect to PEP. At pH 8.0, the same plots were linear with aspartate acting as a competitive inhibitor of PEP. All the other effectors of PEP carboxylase from Salmonella typhimurium and Escherichia coli were found to be ineffective towards the enzyme from T. thiooxidans.

Pyruvate carboxylase of Aspergillus niger: kinetic study of a biotin-containg carboxylase

1969 ◽  
Vol 47 (7) ◽  
pp. 697-710 ◽  
Author(s):  
Helen A. Feir ◽  
Isamu Suzuki
Keyword(s):  
Enzyme Activity ◽  
Aspergillus Niger ◽  
Pyruvate Carboxylase ◽  
Active Sites ◽  
Kinetic Studies ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Ph Optimum ◽  
Metal Cofactor ◽  
Michaelis Constants

Pyruvate carboxylase was partially purified from Aspergillus niger and the properties were studied. The enzyme was found to be cold-labile and protected by 25% glycerol. The pH optimum was determined to be 7.9–8.0. The enzyme was shown to be a biotin-containing enzyme by its inactivation by avidin and protection against such inactivation by biotin. The enzyme activity was stimulated by K+ ions and inhibited by Na+ ions. Acetyl-CoA had no effect on enzyme activity, but L-aspartate was inhibitory. Apparent Michaelis constants were determined for the substrates and metal cofactor involved, i.e. pyruvate, ATP, bicarbonate, and Mg2+.Initial-velocity studies were carried out at varied concentrations of substrates in order to determine the true Michaelis constants and to elucidate the kinetic mechanism of reaction. Product-inhibition studies were carried out with each product (ADP, Pi, and oxalacetate) in combination with every substrate (ATP, bicarbonate, and pyruvate). From these kinetic studies and the existing knowledge on biotin-containing carboxylases, a mechanism was proposed for the action of pyruvate carboxylase which involves three independently active sites on the enzyme, one for each substrate. The interactions between the sites were visualized as being mediated by carboxybiotin formed on the enzyme. A steady-state rate equation was derived that satisfied kinetic results obtained.

Properties of malate dehydrogenase isolated from Methanospirillum hungatii

1979 ◽  
Vol 25 (2) ◽  
pp. 192-200 ◽  
Author(s):  
G. D. Sprott ◽  
R. C. McKellar ◽  
K. M. Shaw ◽  
J. Giroux ◽  
W. G. Martin
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Reaction Mechanism ◽  
Malate Dehydrogenase ◽  
Initial Rate ◽  
Rate Data ◽  
Cell Extracts ◽  
Ping Pong ◽  
Sequential Mechanism ◽  
Variable Substrate

A NADH-linked oxygen-tolerant malate dehydrogenase was purified 270-fold from cell extracts of Methanospirillum hungatii. Inhibitors of the enzyme included ADP, α-ketoglutarate, and excess NADH. Inhibition patterns for ADP were competitive with respect to NADH and non-competitive with respect to oxalacetate. Inhibition by α-ketoglutarate was non-competitive with oxalacetate as variable substrate and uncompetitive with respect to NADH. α-Ketoglutarate is surmised to function as an end-product inhibitor of the enzyme in reactions converting oxalacetate to α-ketoglutarate. No enzyme activity was detected in the direction of malate conversion to oxalacetate, in keeping with a strictly biosynthetic function of the enzyme. An analysis of variance of initial rate data fit to sequential and ping-pong equations showed that a sequential mechanism was preferred. The malate dehydrogenase of M. hungatii resembles those of many other bacteria and eucaryotic cells with respect to molecular weight (61 700) and reaction mechanism, but may be regulated differently.

Serum Cholinesterase Variants: Examination of Several Differential Inhibitors, Salts, and Buffers Used to Measure Enzyme Activity

1971 ◽  
Vol 17 (3) ◽  
pp. 183-191 ◽  
Author(s):  
Philip J Garry
Keyword(s):  
Enzyme Activity ◽  
Sodium Fluoride ◽  
Phosphate Buffer ◽  
Divalent Cations ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Serum Cholinesterase ◽  
Low Concentrations

Abstract Dibucaine, used as a differential inhibitor with acetyl-, propionyl-, and butyrylthiocholine as substrate, clearly identified the "usual" and "atypical" serum cholinesterases. Succinylcholine was also used successfully as a differential inhibitor with butyrylthiocholine as substrate. Sodium fluoride, used as a differential inhibitor, gave conflicting results, depending on whether Tris or phosphate buffer was used in the assay. Mono- and divalent cations (NaCl, KCl, MgCl2, CaCl2, and BaCl2) activated the "usual" and inhibited the "atypical" enzyme at low concentrations. The "usual" enzyme had the same activity in 0.05 mol of Tris or phosphate buffer per liter, while the heterozygous and "atypical" enzymes showed 12 and 42% inhibition, respectively, when assayed in the phosphate buffer. Kinetic studies showed the phosphate acted as a competitive inhibitor of "atypical" enzyme. Km values, determined for "usual" and "atypical" enzymes, were 0.057 and 0.226 mmol/liter, respectively, with butyrylthiocholine as substrate.

scholarly journals Interaction of difluoro-oxaloacetate with aspartate transaminase

1977 ◽  
Vol 161 (2) ◽  
pp. 383-387 ◽  
Author(s):  
P A Briley ◽  
R Eisenthal ◽  
R Harrison ◽  
G D Smith
Keyword(s):  
Steady State ◽  
Competitive Inhibitor ◽  
Aspartate Transaminase ◽  
Steady State Kinetic ◽  
High Concentrations ◽  
State Kinetic ◽  
Uncompetitive Inhibitor

Diffluoro-oxaloacetate behaves as a competitive inhibitor of 2-oxoglutarate and as an uncompetitive inhibitor with respect to aspartate in steady-state kinetic experiments with cytoplasmic aspartate transaminase. In the presence of high concentrations of aspartate transaminase, difluoro-oxaloacetate is slowly transaminated to difluoro-aspartate, suggesting its use as a kinetic probe to study the reactions of the aminic form of the enzyme.

Receptor kinetics pertaining to blockade of nerve-released transmitter at synapses

1988 ◽  
Vol 233 (1273) ◽  
pp. 461-475 ◽  
Keyword(s):  
Channel Blocker ◽  
Competitive Inhibitor ◽  
Tissue Response ◽  
Receptor Density ◽  
Receptor Kinetics ◽  
Tissue Responses ◽  
Competitive Inhibitors ◽  
Transmitter Substance ◽  
Uncompetitive Inhibitor ◽  
Temporal Inhomogeneity

The question is raised as to whether competitive inhibitors should block responses of tissue to nerve-released neurotransmitter to the same extent as they block equivalent responses to exogenous agonist. From a simple dynamic model of synaptic events, which takes into account non-constancy of transmitter concentration in space and time, it is deduced that equal blockade of responses to nerve-released and exogenous transmitter substance will occur if: (i) there are locally many more receptor molecules than transmitter molecules; (ii) the active agonist–receptor complex, A n R, has n = 1 ; and (iii) tissue response is insensitive to spatial or temporal inhomogeneity of AR. In such a case there will also be equal sensitivity of responses to other modes of inhibition: irreversible competitive, uncompetitive, and non-competitive. Equal blockade of responses to equi-effective endogenous and exogenous agonist will also occur if nerve stimulation gives rise to a steady uniform concentration of agonist, so that equilibrium kinetics are applicable. When n > 1 and/or when tissue responses reflect local peak A n R, response to nerve-released transmitter will be relatively insensitive to receptor blockade by a competitive inhibitor. The same is true for irreversible competitive blockade or for modulation of receptor density. However, an uncompetitive inhibitor (e. g. a ‘channel blocker’) may be more effective against nerve-released agonist than against exogenous agonist.

scholarly journals Mucin biosynthesis. Properties of a bovine tracheal mucin β-6-N-acetylglucosaminyltransferase

1985 ◽  
Vol 227 (2) ◽  
pp. 405-412 ◽  
Author(s):  
P W Cheng ◽  
W E Wingert ◽  
M R Little ◽  
R Wei
Keyword(s):  
Enzyme Activity ◽  
Freezing Point ◽  
Cetylpyridinium Chloride ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Gas Chromatography Mass Spectrometry ◽  
Tween 20 ◽  
Group A ◽  
Michaelis Constants ◽  
Triton X

We have characterized a bovine tracheal mucin beta-6-N-acetylglucosaminyltransferase that catalyses the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the C-6 of the N-acetylgalactosamine residue of galactosyl-β 1→3-N-acetylgalactosamine. Optimal enzyme activity was obtained between pH 7.5-8.5, at 5mM-MnCl2, and at 0.06-0.08% (v/v) Triton X-100 (or Nonidet P-40), or 0.5-5.0% (v/v) Tween 20. Ba2+, Mg2+ and Ca2+ could partially replace Mn2+, but Co2+, Fe2+, Cd2+ and Zn2+ could not. Sodium dodecyl sulphate, cetylpyridinium chloride, sodium deoxycholate, octyl beta-D-glucoside, digitonin and alkyl alcohols were less effective in enhancing enzyme activity, and dimethyl sulphoxide was ineffective. The apparent Michaelis constants were 1.25 mM for UDP-N-acetylglucosamine, 0.94-3.34 mM for freezing-point-depressing glycoprotein and 0.19 mM for periodate-treated blood-group-A porcine submaxillary mucin. Asialo ovine submaxillary mucin could not serve as the glycosyl acceptor. The structure of the 14C-labelled oligosaccharide obtained by alkaline-borohydride treatment of the product was identified as Gal beta 1→3(Glc-NAc beta 1→6)N-acetylgalactosaminitol by beta-hexosaminidase treatment, gas chromatography-mass spectrometry and 1H-n.m.r. (270 MHz) analysis. The enzyme is important in the regulation of mucin oligosaccharide biosynthesis.

scholarly journals Evidence for the importance of arginine residues in pig kidney alkaline phosphatase

1979 ◽  
Vol 181 (1) ◽  
pp. 137-142 ◽  
Author(s):  
M N Woodroofe ◽  
P J Butterworth
Keyword(s):  
Alkaline Phosphatase ◽  
Active Site ◽  
Competitive Inhibitor ◽  
Arginine Residue ◽  
Pig Kidney ◽  
Close Proximity ◽  
Arginine Residues ◽  
Km Value ◽  
Uncompetitive Inhibitor ◽  
Kidney Alkaline Phosphatase

The arginine-specific reagents 2,3-butanedione and phenylglyoxal inactivate pig kidney alkaline phosphatase. As inactivation proceeds there is a progressive fall in Vmax. of the enzyme, but no demonstrable change in the Km value for substrate. Pi, a competitive inhibitor, and AMP, a substrate of the enzyme, protect alkaline phosphatase against the arginine-specific reagents. These effects are explicable by the assumption that the enzyme contains an essential arginine residue at the active site. Protection is also afforded by the uncompetitive inhibitor NADH through a partially competive action against the reagents. Enzyme that has been exposed to the reagents has a decreased sensitivity to NADH inhibition. It is suggested that an arginine residue is important for NADH binding also, although this residue is distinct from that at the catalytic site. The protection given by NADH against loss of activity is indicative of the close proximity of the active and NADH sites.

scholarly journals Novel Substrates for Kinases Involved in the Biosynthesis of Inositol Pyrophosphates and Their Enhancement of ATPase Activity of a Kinase

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3601
Author(s):  
Raja Mohanrao ◽  
Ruth Manorama ◽  
Shubhra Ganguli ◽  
Mithun C. Madhusudhanan ◽  
Rashna Bhandari ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Catalytic Domain ◽  
Inositol Phosphate ◽  
Substrate Recognition ◽  
Competitive Inhibitor ◽  
Inositol Polyphosphates ◽  
Inositol Pyrophosphate ◽  
Phosphate Donor ◽  
Inositol Pyrophosphates

IP6K and PPIP5K are two kinases involved in the synthesis of inositol pyrophosphates. Synthetic analogs or mimics are necessary to understand the substrate specificity of these enzymes and to find molecules that can alter inositol pyrophosphate synthesis. In this context, we synthesized four scyllo-inositol polyphosphates—scyllo-IP5, scyllo-IP6, scyllo-IP7 and Bz-scyllo-IP5—from myo-inositol and studied their activity as substrates for mouse IP6K1 and the catalytic domain of VIP1, the budding yeast variant of PPIP5K. We incubated these scyllo-inositol polyphosphates with these kinases and ATP as the phosphate donor. We tracked enzyme activity by measuring the amount of radiolabeled scyllo-inositol pyrophosphate product formed and the amount of ATP consumed. All scyllo-inositol polyphosphates are substrates for both the kinases but they are weaker than the corresponding myo-inositol phosphate. Our study reveals the importance of axial-hydroxyl/phosphate for IP6K1 substrate recognition. We found that all these derivatives enhance the ATPase activity of VIP1. We found very weak ligand-induced ATPase activity for IP6K1. Benzoyl-scyllo-IP5 was the most potent ligand to induce IP6K1 ATPase activity despite being a weak substrate. This compound could have potential as a competitive inhibitor.

HYPERAMMONEMIA DUE TO A MUTANT ENZYME OF ORNITHINE TRANSCARBAMYLASE

PEDIATRICS ◽  
1971 ◽  
Vol 48 (4) ◽  
pp. 595-600
Author(s):  
Ichiro Matsuda ◽  
Shinichiro Arashima ◽  
Haruo Nambu ◽  
Yasuo Takekoshi ◽  
Michiya Anakura
Keyword(s):  
In Vitro Study ◽  
Ornithine Transcarbamylase ◽  
Vitro Study ◽  
Mutant Enzyme ◽  
Elevated Blood ◽  
Blood Ammonia ◽  
Carbamyl Phosphate

In a case of congenital hyperammonemia described in an 8½-month-old girl, elevated blood ammonia was shown to result from a mutant enzyme of ornithine transcarbamylase. An in vitro study indicated a decrease in affinity of the enzyme for carbamyl phosphate but not for ornithine.

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