scholarly journals The mechanism of phosphoglucomutase from Micrococcus lysodeikticus

1974 ◽  
Vol 137 (3) ◽  
pp. 453-461 ◽  
Author(s):  
John B. Clarke ◽  
Hubert G. Britton
Keyword(s):  
Quantitative Agreement ◽  
Rabbit Muscle ◽  
Muscle Enzyme ◽  
Previous Evidence ◽  
Micrococcus Lysodeikticus ◽  
Sequential Mechanism ◽  
Diphosphate Glucose ◽  
High Concentrations ◽  
Intramolecular Transfer ◽  
Substrate Concentrations

The mechanism of the phosphoglucomutase from Micrococcus lysodeikticus was investigated. Induced-transport tests at low substrate concentrations (0.15mm) showed co-transport of the 32P label but no induced transport of the 14C label, which is in quantitative agreement with a phosphoenzyme mechanism with a rapid isomerization of the phosphoenzyme. The results excluded an intramolecular transfer of phosphate and could only have been compatible with a sequential mechanism if the Km for glucose 1-phosphate had been over 20 times smaller than the measured value. The results of induced-transport tests at intermediate concentrations (1mm) with both labels agreed quantitatively with a phosphoenzyme mechanism, and induced-transport tests with 14C-labelled substrates at high concentrations (26mm) indicated that the rate constants for isomerization of the phosphoenzyme must be greater than about 3×106s-1. Consistent with these findings is the fact that 14C label exchanged between the substrates twice as rapidly as the 32P label at chemical equilibrium. Further, since the 14C label exchanged between the substrates about ten times more rapidly than between the substrates and glucose 1,6-diphosphate, glucose 1,6-diphosphate is not an obligatory intermediate in the interconversion of the substrates. It is concluded that, contrary to previous evidence, the mechanism of the enzyme from M. lysodeikticus is essentially that of the rabbit muscle enzyme. To account for the rapid isomerization of the phosphoenzyme in both cases a mechanism is proposed in which there is no formal isomerization of the phosphoenzyme.

scholarly journals Mechanism of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutase from rabbit muscle

1972 ◽  
Vol 130 (2) ◽  
pp. 397-410 ◽  
Author(s):  
H. G. Britton ◽  
J. B. Clarke
Keyword(s):  
Chemical Equilibrium ◽  
Phosphoglycerate Mutase ◽  
Rabbit Muscle ◽  
Kinetic Measurements ◽  
Rate Limiting Step ◽  
Ping Pong ◽  
Sequential Mechanism ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Intramolecular Transfer

1. The properties and kinetics of the 2,3-diphosphoglycerate-dependent phosphoglycerate mutases are discussed. There are at least three possible mechanisms for the reaction: (i) a phosphoenzyme (Ping Pong) mechanism; (ii) an intermolecular transfer of phosphate from 2,3-diphosphoglycerate to the substrates (sequential mechanism); (iii) an intramolecular transfer of phosphate. It is concluded that these mechanisms cannot be distinguished by conventional kinetic measurements. 2. The fluxes for the different mechanisms are calculated and it is shown that it should be possible to distinguish between the mechanisms by appropriate induced-transport tests and by comparing the fluxes of 32P- and 14C-labelled substrates at chemical equilibrium. 3. With 14C-labelled substrates no induced transport was found over a wide concentration range, and with 32P-labelled substrates co-transport occurred that was independent of concentration over a twofold range. 14C-labelled substrates exchange at twice the rate of 32P-labelled substrates at chemical equilibrium. The results were completely in accord with a phosphoenzyme mechanism and indicated a rate constant for the isomerization of the phosphoenzyme of not less than 4×106s−1. The intramolecular transfer of phosphate (and intermolecular transfer between two or more molecules of substrate) were completely excluded. The intermolecular transfer of phosphate from 2,3-diphosphoglycerate would have been compatible with the results only if the Km for 2-phosphoglycerate had been over 7.5-fold smaller than the observed value and if an isomerization of the enzyme-2,3-diphosphoglycerate complex had been the major rate-limiting step in the reaction. 4. The very rapid isomerization of the phosphoenzyme that the experiments demonstrate suggests a mechanism that does not involve a formal isomerization. According to this new scheme the enzyme is closely related mechanistically and perhaps evolutionarily to a 2,3-diphosphoglycerate diphosphatase.

scholarly journals Properties and reaction with iodoacetamide of adenosine 5′-triphosphate–creatinine phosphotransferase from human skeletal muscle. Further evidence about the role of the essential thiol group in relation to the mechanism of action

1970 ◽  
Vol 117 (3) ◽  
pp. 513-523 ◽  
Author(s):  
I. Kumudavalli ◽  
B. H. Moreland ◽  
D. C. Watts
Keyword(s):  
Skeletal Muscle ◽  
Transition State ◽  
Thiol Group ◽  
Equilibrium Mixture ◽  
Common Mechanism ◽  
Kinetic Properties ◽  
Rabbit Muscle ◽  
Muscle Enzyme ◽  
Similar Chemical ◽  
High Concentrations

1. The purification of creatine kinase from human and monkey skeletal muscle by horizontal electrophoresis on Sephadex blocks is described. 2. The purified enzymes are shown to have similar chemical and kinetic properties to the rabbit muscle enzyme and a common mechanism is inferred. 3. Iodoacetamide has a similar apparent second-order inhibition constant with the human and rabbit enzymes, but the inhibition does not go to completion with the former. This is even more marked with the monkey enzyme, which has more reactive thiol groups, but inhibition is only about 50%. 4. Single substrates have little effect on the inhibition by iodoacetamide, but with the primate enzymes, in contrast with the rabbit enzyme, high concentrations of ADP–Mg2+ plus creatine convert the essential thiol group from being pH-independent into one with a normal ionization. Low concentrations of ADP–Mg2+ plus creatine first enhance the rate of inactivation, but cause protection as the reaction proceeds. These results are interpreted to indicate an activation of the thiol group on the subunit to which the substrates bind and a co-operatively induced decrease in the activity of the thiol group on the other subunit which lacks substrates. 5. The effects of a substrate equilibrium mixture on the rate of inhibition are essentially those of ADP–Mg2+ plus creatine. 6. Since no substrate combination affords significant protection to the thiol group associated with the catalytic site to which the substrates are bound, it is concluded that any mechanism involving the thiol group in a direct participation in the transition-state complex of the catalytic reaction must be abandoned unless the transition state is only a small part of the time taken for one catalytic cycle.

scholarly journals Homogeneous Enzyme Immunoassay for Triiodothyronine in Serum

2001 ◽  
Vol 47 (3) ◽  
pp. 569-574 ◽  
Author(s):  
Christina D Karapitta ◽  
Theodore G Sotiroudis ◽  
Athanassios Papadimitriou ◽  
Aristotelis Xenakis
Keyword(s):  
Enzyme Immunoassay ◽  
Glycogen Phosphorylase ◽  
Serum Proteins ◽  
Rabbit Muscle ◽  
Glycogen Phosphorylase B ◽  
Low Concentrations ◽  
Enzyme Conjugate ◽  
High Concentrations ◽  
Treatment Step ◽  
Homogeneous Enzyme Immunoassay

Abstract Background: The concentration of triiodothyronine (T3) in human serum is extremely low and can be determined only by very sensitive methods. We developed a homogeneous enzyme immunoassay for T3 analysis in unextracted serum. Methods: A T3 derivative was conjugated to the −SH groups of glycogen phosphorylase b (GPb) from rabbit muscle. Conjugation caused inhibition of enzyme activity, and the enzyme conjugate was reactivated upon binding of anti-T3 antibody. Activation was blocked by the presence of non-antibody-bound T3; this was the basis for the development of the homogeneous enzyme immunoassay for T3 by determining GPb activity fluorometrically. Results: We used furosemide to block the interaction of T3 with serum proteins with T3-binding sites, avoiding any serum treatment step. T3 was measured in the range 0.3–8 μg/L. T3 values obtained by this assay correlated well with those obtained by a RIA (y = 0.97x − 0.07 μg/L; r = 0.96; n = 92). Within- and between-run imprecision (CV) was 5–9% for normal and high concentrations and 16–20% for low concentrations. Conclusions: Chemical modification of GPb with a T3 derivative allows the development of a simple homogeneous enzyme immunoassay for T3 in unextracted serum.

Detection of inhibitors in reduced nicotinamide adenine dinucleotide by kinetic methods.

1976 ◽  
Vol 22 (10) ◽  
pp. 1648-1654 ◽  
Author(s):  
B F Howell ◽  
S Margolis ◽  
R Schaffer
Keyword(s):  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine ◽  
Bicarbonate Buffer ◽  
Kinetic Methods ◽  
Pig Muscle ◽  
High Concentrations ◽  
Ph Buffer ◽  
Reduced Nicotinamide Adenine Dinucleotide ◽  
Substrate Concentrations ◽  
Curve Upward

Abstract Methods are described for detection of lactate dehydrogenase (LDH) inhibitors in preparations of reduced nicotinamide adenine dinucleotide. They are (a) comparison of values by kinetic methods with those measured for highly purified NADH and (b) examination of Lineweaver-Burk plots. Chromatographic inhomogeneities are correlated with deviant values for the kinetic constants of NADH preparations. Lineweaver-Burk plots that curve upward at the high concentrations or have a larger or smaller than normal slope may indicate the presence of inhibitor. As determined in bicarbonate buffer (0.11 mol/liter, pH 7.9) by use of 0.600 mmol/liter pyruvate and NADH freshly separated from impurities by chromatography on diethyl-aminoethyl-cellulose, the Km (apparent) of NADH at 25 degrees C has the value 8.11 +/- 0.71 mumol/liter (SD, n = 28) with LDH-1 (pig heart, 2.48 +/- 0.05 U per milliliter of reaction mixture, or 41.3 +/- 0.8 nmol/liter per second). Under similar conditions, the Km (apparent) of NADH has the value of 8.57 +/- 1.58 mol/liter (SD, n = 21) with LDH-5 (pig muscle, 1.77 +/- 0.03 U/ml of reaction mixture), or 29.4 +/- 0.6 nmol/liter per second). At infinite substrate concentrations with the same pH, buffer, and temperature, the Km (apparent) for NADH was 26.0 +/- 0.63 mumol/liter with LDH-1 and 23.2 +/- 4.6 mumol/liter with LDH-5.

The Carboxy-Terminal Region of Sturgeon Muscle Aldolase

1971 ◽  
Vol 49 (3) ◽  
pp. 372-375
Author(s):  
P. J. Anderson
Keyword(s):  
Amino Acids ◽  
Terminal Region ◽  
Rabbit Muscle ◽  
Muscle Enzyme ◽  
Molar Ratios ◽  
Carboxy Terminal Region ◽  
Kinetic Effects ◽  
Carboxy Terminal ◽  
Similar Kinetic

The modification of the carboxy-terminal of sturgeon muscle aldolase with carboxypeptidase produces similar kinetic effects to those observed on modification of the rabbit muscle enzyme. The nature and molar ratios of amino acids released indicate that differences between the two enzymes occur in the carboxy-terminal region.

scholarly journals Folding domains and intramolecular ionic interactions of lysine residues in glyceraldehyde 3-phosphate dehydrogenase

1977 ◽  
Vol 161 (1) ◽  
pp. 49-62 ◽  
Author(s):  
J M Lambert ◽  
R N Perham
Keyword(s):  
Catalytic Activity ◽  
Aspartic Acid ◽  
Three Dimensional ◽  
Ion Pair ◽  
Dimensional Structure ◽  
Ionic Interactions ◽  
Rabbit Muscle ◽  
Amino Groups ◽  
Muscle Enzyme ◽  
Lysine Residues

1. Treatment with methyl acetimidate was used to probe the topography of several tetrameric glyceraldehyde 3-phosphate dehydrogenases, in particular the holoenzymes from rabbit muscle and Bacillus stearothermophilus. During the course of the reaction with the rabbit muscle enzyme, the number of amino groups fell rapidly from the starting value of 27 per subunit to a value of approx. five per subunit. This number could be lowered further to values between one and two per subunit by a second treatment with methyl acetimidate. The enzyme remained tetrameric throughout and retained 50% of its initial catalytic activity at the end of the experiment. 2. Use of methyl [1-14C]acetimidate and small-scale methods of protein chemistry showed that only one amino group per subunit, that of lysine-306, was completely unavailable for reaction with imido ester in the native enzyme. This results is consistent with the structure of the highly homologous glyceraldehyde 3-phosphate dehydrogenase of lobster muscle deduced from X-ray-crystallographic analysis, since lysine-306 can be seen to form an intrachain ion-pair with aspartic acid-241 in the hydrophobic environment of a subunit-subunit interface. 3. Several other amino groups in the rabbit muscle enzyme that reacted only slowly with the reagent were also identified chemically. These were found to be located entirely in the C-terminal half of the polypeptides chain, which comprises a folding domain associated with catalytic activity and subunit contact in the three-dimensional structure. Slow reaction of these ‘surface’ amino groups with methyl acetimidate is attributed to intramolecular ionic interactions of the amino groups with neighbouring side-chain carboxyl groups, a conclusion that is compatible with the reported three-dimensional structure and with the dependence of the reaction of ionic stength. 4. Very similar results were obtained with the enzymes from B. stearothermophilus and from ox muscle and ox liver, supporting the view that the ion-pair involving lysine-306 and aspartic acid-241 will be a common structural feature in glyceraldehyde-3-phosphate dehydrogenases. The B. stearothermophilus enzyme was fully active after modification. 5. No differences could be detected between the enzymes from ox muscle and ox liver, in accord with other evidence that points to the identify of these enzymes. 6. The pattern of slowly reacting amino groups in the enzyme from B. stearothermophilus, although similar to that of the mammalian enzymes, indicated one or two additional intramolecular ionic interactions of lysine residues that might contribute to the thermal stability of this enzyme.

scholarly journals Continuous enzymatic stirred tank reactor cascade with unconventional medium yielding high concentrations of (S)-2-hydroxyphenyl propanone and its derivatives

2021 ◽  
Author(s):  
Reinhard Oeggl ◽  
Juliane Diedrich ◽  
Eric von Lieres ◽  
Dörte Rother
Keyword(s):  
Green Chemistry ◽  
Flow Chemistry ◽  
Stirred Tank ◽  
Stirred Tank Reactor ◽  
Tank Reactor ◽  
High Substrate ◽  
High Concentrations ◽  
Substrate Concentrations

The implementation of biocatalysis in flow chemistry offers synergistic synthesis advantages in line with green chemistry principles. Yet, the conversion of high substrate concentrations is most often hindered by substrate...

Ultrastructural localization with Transmission Electron Microscopy of the catalytic activity of adenosine monophosphate deaminase in murine skeletal muscle

1991 ◽  
Vol 49 ◽  
pp. 280-281
Author(s):  
K. Bielat ◽  
G. Tritsch
Keyword(s):  
Adenosine Deaminase ◽  
Adenosine Monophosphate ◽  
Ultrastructural Localization ◽  
Rabbit Muscle ◽  
Amp Deaminase ◽  
Muscle Enzyme ◽  
Transmission Electron ◽  
Adenosine Deaminase Activity ◽  
Murine Skeletal Muscle ◽  
Excised Tissue

AMP deaminase was visualized in a manner analogous to our previous approach with adenosine deaminase. The chloro-analog of AMP, i.e., 6 Chloropurine riboside 5’ monophosphate (CPRMP) (from Sigma) was shown to be a substrate of this enzyme which liberates Cl- is precipitate with added Ag+, electron dense Ag° grains are deposited at loci of enzyme activity. The substrate at a concentration of 1.1 mM in 50 mM HEPES buffer, pH 7.2, in the presence of 150 mM K* (as the acetate), 3mM ATP and 10μM pentostatin (deoxycoformycin) was incubated with freshly excised tissue from a female C-57 BL/6 mouse. The substrate concentration is 1.4 times Km for rabbit muscle enzyme (Sigma), and K+ and ATP are allosteric activators of this enzyme. Because many cells have ectonucleotidase activity, pentostatin was added to prevent manifestation of adenosine deaminase activity of dechlorination of any nucleoside that might be formed.

The catalytic activity of adenosine monophosphate deaminase in murine thymus as revealed by Transmission Electron Microscopy

1994 ◽  
Vol 52 ◽  
pp. 186-187
Author(s):  
K. Bielat ◽  
G. Tritsch
Keyword(s):  
Electron Microscopy ◽  
Adenosine Deaminase ◽  
Adenosine Monophosphate ◽  
Rabbit Muscle ◽  
Amp Deaminase ◽  
Nucleotidase Activity ◽  
Previous Approach ◽  
Muscle Enzyme ◽  
Transmission Electron ◽  
Excised Tissue

AMP deaminase was visualized in a manner analogous to our previous approach with adenosine deaminase. The caloro-analog of AMP, i.e., 6 Chloropurine riboside 5′ monophosphate (CPRMP) (from Sigma) was shown to be a substrate of this enzyme which liberates Cl− which is precipitated with added Ag+, and, after exposure to light electron sense Ag° grains are deposited at loci of enzyme activity. The substrate at a concentration of 1.1 mM in 50 mM HEPES buffer, pH 7.2, in the presence of 150 mM K+ (as the acetate), 3mM ATP and 10μM pentostatin deoxycoformycin) was incubated with freshly excised tissue from a female C-57 BL/6 mouse. The substrate concentration is 1.4 times Km for rabbit muscle enzyme (Sigma), and K+ and ATP are allosteric activators of this Enzyme. Because many cells contain ecto 5′-nucleotidase activity, pentostatin was added to prevent manifestation of denosine deaminase activity of dechlorination of any nucleoside that might be formed. Any Cl− formed by adenosine deaminase would be precipitated with the added Ag+ and thus result in Ago deposits which would be indistinguishable from the Ago deposited by AMP deaminase.

The catalytic activity of adenosine monophosphate deaminase in murine duodenum as revealed by transmission electron microscopy

1993 ◽  
Vol 51 ◽  
pp. 302-303
Author(s):  
K. Bielat ◽  
G. Tritsch
Keyword(s):  
Adenosine Deaminase ◽  
Adenosine Monophosphate ◽  
Rabbit Muscle ◽  
Amp Deaminase ◽  
Nucleotidase Activity ◽  
Previous Approach ◽  
Muscle Enzyme ◽  
Transmission Electron ◽  
Adenosine Deaminase Activity ◽  
Excised Tissue

AMP deaminase was visualized in a manner analogous to our previous approach with adenosine deaminase. The chloro-analog of AMP, i.e., 6 Chloropurine riboside 5' monophosphate (CPRMP) (from Sigma) was shown to be a substrate of this enzyme which liberates Cl- which is precipitated with added Ag+, and, after exposure to light electron dense Ag° grains are deposited at loci of enzyme activity. The substrate at a concentration of 1.1 mM in 50 mM HEPES buffer, pH 7.2, in the presence of 150 mM K+ (as the acetate), 3mM ATP and 10¼M pentostathi (deoxycoformycin) was incubated with freshly excised tissue from a female C-57 BL/6 mouse. The substrate concentration is 1.4 times Km for rabbit muscle enzyme (Sigma), and K+ and ATP are allosteric activators of this enzyme. Because many cells have ecto 5'-nucleotidase activity, pentostathi was added to prevent manifestation of adenosine deaminase activity of dechlorination of any nucleoside that might be formed.

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