scholarly journals Proton Exchange of the Pro-S Hydrogen at C-1 in Dihydroxyacetone Phosphate D-Fructose 1, 6-Bisphosphate and d-Fructosehate, d-Fructose 1,6-Bisphosphate and Catalysed by Rabbit-Muscle Aldolase

1976 ◽  
Vol 66 (1) ◽  
pp. 95-104 ◽  
Author(s):  
Gordon LOWE ◽  
Rex F. PRATT
Keyword(s):  
Proton Exchange ◽  
Dihydroxyacetone Phosphate ◽  
Rabbit Muscle ◽  
Fructose 1,6 Bisphosphate

scholarly journals Fructose 1,6-bisphosphate aldolase from rabbit muscle. The isomerization of the enzyme-dihydroxyacetone phosphate complex

1977 ◽  
Vol 167 (2) ◽  
pp. 361-366 ◽  
Author(s):  
E Grazi ◽  
M Blanzieri
Keyword(s):  
Competitive Inhibitor ◽  
Dihydroxyacetone Phosphate ◽  
Rabbit Muscle ◽  
Keto Form ◽  
First Order ◽  
Phosphate Complex ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Dead End ◽  
Fructose 1,6 Bisphosphate

The formation and dissociation of the aldolase-dihydroxyacetone phosphate complex were studied by following changes in A240 [Topper, Mehler & Bloom (1957), Science 126, 1287-1289]. It was shown that the enzyme-substrate complex (ES) slowly isomerizes according to the following reaction: (formula: see text) the two first-order rate constants for the isomerization step being k+2 = 1.3s-1 and k-2 = 0.7s-1 at 20 degrees C and pH 7.5. The dissociation of the ES complex was provoked by the addition of the competitive inhibitor hexitol 1,6-bisphosphate. At 20 degrees C and pH 7.5, k+1 was 4.7 X 10(6)M-1-S-1 and k-1 was 30s-1. Both the ES and the ES* complexes react rapidly with 1.7 mM-glyceraldehyde 3-phosphate, the reaction being practically complete in 40 ms. This shows that the ES* complex is not a dead-end complex. Evidence was also provided that aldolase binds and utilizes only the keto form of dihydroxyacetone phosphate.

scholarly journals A new intermediate of the aldolase reaction, the pyruvaldehyde-aldolase-orthophosphate complex

1978 ◽  
Vol 175 (2) ◽  
pp. 361-365 ◽  
Author(s):  
E Grazi ◽  
G Trombetta
Keyword(s):  
Rate Constant ◽  
Order Rate Constant ◽  
Dihydroxyacetone Phosphate ◽  
Rabbit Muscle ◽  
Spontaneous Release ◽  
First Order ◽  
Neutral Ph ◽  
Order Rate ◽  
Two Phases ◽  
Fructose 1,6 Bisphosphate

Fructose 1,6-bisphosphate aldolase from rabbit muscle forms by reaction with dihydroxyacetone phosphate a pyruvaldehyde-aldolase-orthophosphate complex that is in equilibrium with the eneamine intermediate. The new intermediate accumulates in two phases. The first one is practically complete in 40ms, and the second occurs with an apparent first-order rate constant of 4.6 +/- 0.5s-1. The new intermediate breaks down slowly with the release into the medium of pyruvaldehyde and Pi. The rate of the spontaneous release is higher at acidic than at neutral pH.

scholarly journals The proton exchange of the pro-S hydrogen atom at C-1 in dihydroxyacetone phosphate and d-fructose 1,6-bisphosphate catalysed by class-I and class-II aldolases

1978 ◽  
Vol 171 (3) ◽  
pp. 539-542 ◽  
Author(s):  
A Galdes ◽  
H A O Hill
Keyword(s):  
Hydrogen Atom ◽  
Class Ii ◽  
Proton Exchange ◽  
Class I ◽  
Dihydroxyacetone Phosphate ◽  
Fructose 1,6 Bisphosphate

The efficacy of class-I and class-II aldolases in catalysing the C-1 proton exchange in fructose 1,6-bisphosphate and dihydroxyacetone phosphate was investigated. The rate of this reaction was at least two orders of magnitude slower in class-II than in the class-I aldolases. It is suggested that this difference reflects the formation of different intermediates in the reactions catalysed by the two classes of aldolase.

scholarly journals Triosephosphate isomerase deficiency: consequences of an inherited mutation at mRNA, protein and metabolic levels

2005 ◽  
Vol 392 (3) ◽  
pp. 675-683 ◽  
Author(s):  
Judit Oláh ◽  
Ferenc Orosz ◽  
László G. Puskás ◽  
László Hackler ◽  
Margit Horányi ◽  
...  
Keyword(s):  
Steady State ◽  
Triosephosphate Isomerase ◽  
Specific Activity ◽  
Energy State ◽  
Dihydroxyacetone Phosphate ◽  
Peptidase Activity ◽  
Mrna Levels ◽  
Regulatory Enzymes ◽  
Computational Data ◽  
Fructose 1,6 Bisphosphate

Triosephosphate isomerase (TPI) deficiency is a unique glycolytic enzymopathy coupled with neurodegeneration. Two Hungarian compound heterozygote brothers inherited the same TPI mutations (F240L and E145Stop), but only the younger one suffers from neurodegeneration. In the present study, we determined the kinetic parameters of key glycolytic enzymes including the mutant TPI for rational modelling of erythrocyte glycolysis. We found that a low TPI activity in the mutant cells (lower than predicted from the protein level and specific activity of the purified recombinant enzyme) is coupled with an increase in the activities of glycolytic kinases. The modelling rendered it possible to establish the steady-state flux of the glycolysis and metabolite concentrations, which was not possible experimentally due to the inactivation of the mutant TPI and other enzymes during the pre-steady state. Our results showed that the flux was 2.5-fold higher and the concentration of DHAP (dihydroxyacetone phosphate) and fructose 1,6-bisphosphate increased 40- and 5-fold respectively in the erythrocytes of the patient compared with the control. Although the rapid equilibration of triosephosphates is not achieved, the energy state of the cells is not ‘sick’ due to the activation of key regulatory enzymes. In lymphocytes of the two brothers, the TPI activity was also lower (20%) than that of controls; however, the remaining activity was high enough to maintain the rapid equilibration of triosephosphates; consequently, no accumulation of DHAP occurs, as judged by our experimental and computational data. Interestingly, we found significant differences in the mRNA levels of the brothers for TPI and some other, apparently unrelated, proteins. One of them is the prolyl oligopeptidase, the activity decrease of which has been reported in well-characterized neurodegenerative diseases. We found that the peptidase activity of the affected brother was reduced by 30% compared with that of his neurologically intact brother.

Triosephosphate metabolism by mature boar spermatozoa

1997 ◽  
Vol 9 (6) ◽  
pp. 577 ◽  
Author(s):  
A. R. Jones
Keyword(s):  
Glycolytic Pathway ◽  
Dihydroxyacetone Phosphate ◽  
Glycolytic Intermediates ◽  
Boar Sperm ◽  
Equilibrium Concentrations ◽  
Fructose 1,6 Bisphosphate ◽  
Boar Spermatozoa

Boar sperm rapidly interconverted dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, produced fructose-1,6-bisphosphate, approximately equilibrium concentrations of fructose 6-phosphate and glucose 6-phosphate but not glycerol or glycerol 3-phosphate. In the presence of 3-chloro-1-hydroxypropanone, an inhibitor of stage 2 of the glycolytic pathway, the triosephosphates were metabolized faster, produced less fructose-1,6-bisphosphate, fructose 6-phosphate and glucose 6-phosphate, but not glycerol or glycerol 3-phosphate. This suggests that these cells may have the capacity to convert glycolytic intermediates into a storage metabolite to conserve carbon atoms for the eventual synthesis of lactate.

scholarly journals Erythrocyte glycolysis and its marked alterations by muscular exercise in type VII glycogenosis

Blood ◽  
1988 ◽  
Vol 71 (4) ◽  
pp. 1130-1134 ◽  
Author(s):  
T Shimizu ◽  
N Kono ◽  
H Kiyokawa ◽  
Y Yamada ◽  
N Hara ◽  
...  
Keyword(s):  
Bed Rest ◽  
Normal Subjects ◽  
Dihydroxyacetone Phosphate ◽  
Crossover Point ◽  
Control Value ◽  
Myogenic Factors ◽  
Fructose 1,6 Bisphosphate ◽  
2,3 Dpg ◽  
Ergometric Exercise

Abstract Levels of erythrocyte glycolytic intermediates after the phosphofructokinase (PFK) step, including 2,3-bisphosphoglycerate (2,3- DPG), were decreased at rest in patients from separate families with type VII glycogenosis. The concentration of 2,3-DPG was about half of the normal control value during a period of unrestricted daily activity but was further decreased to one third of normal after a one-day bed rest. Mild ergometric exercise rapidly increased the levels of fructose- 1,6-bisphosphate, dihydroxyacetone phosphate plus glyceraldehyde-3- phosphate, and 2,3-DPG in patients' circulating erythrocytes but did not in those of normal subjects. This indicated that a crossover point at the PFK step in glycolysis disappeared after physical exercise and, consequently, the 2,3-DPG concentration, which had decreased because of blockage of the PFK step, was restored considerably. This apparently exercise-related alteration in intermediary metabolism at the beginning of glycolysis was reproduced in vitro by incubating normal erythrocytes in the presence of inosine or ammonia, both of which have increased levels in circulating blood during and after exercise in this disorder. We conclude that physical activity in addition to a genetic deficiency in erythrocyte PFK affects glycolysis in erythrocytes in type VII glycogenosis and that myogenic factors released from exercising muscles may be responsible for this change.

FRUCTOSE DIPHOSPHATE ALDOLASE FROM FUSARIUM OXYSPORUM f. LYGOPERSICI: II. CHARACTERISTICS OF THE EXCHANGE REACTION

1967 ◽  
Vol 45 (10) ◽  
pp. 1505-1511 ◽  
Author(s):  
J. M. Ingram
Keyword(s):  
Methylene Blue ◽  
Fusarium Oxysporum ◽  
Exchange Reaction ◽  
Active Site ◽  
Reaction Rates ◽  
Exchange Capacity ◽  
Proton Exchange ◽  
Dihydroxyacetone Phosphate ◽  
Cleavage Reaction ◽  
Twofold Decrease

Fruetose-1,6-diphosphate aldolase purified from extracts of Fusarium oxysporum f. lycopersici was examined in detail. The different pH optima obtained for the proton exchange (pH 6.0) and cleavage (pH 7.5) reactions indicate that this enzyme resembles the yeast FDP-aldolase rather than the aldolase from muscle. When the enzyme was dialyzed extensively, in the absence of Cleland's reagent, it was found that neither the cleavage nor the exchange reaction rates were increased appreciably after addition of mercaptan. Treatment of the enzyme with either methylene blue in the presence of light or with diazosulfanilic acid, two known inhibitors of the cleavage reaction, resulted in almost complete inhibition of FDP cleavage but only a twofold decrease in the exchange capacity. These results substantiate our previous findings that the inhibitors react with residues involved in the D-glyceraldehyde-3-phosphate rather than the dihydroxyacetone phosphate active site.

KINETIC STUDIES ON THE MECHANISM OF CYTOPLASMIC L-α-GLYCEROPHOSPHATE DEHYDROGENASE OF RABBIT SKELETAL MUSCLE

1966 ◽  
Vol 44 (10) ◽  
pp. 1301-1317 ◽  
Author(s):  
William J. Black
Keyword(s):  
Kinetic Data ◽  
Substrate Inhibition ◽  
Product Inhibition ◽  
Reduced Form ◽  
Ultraviolet Absorption Spectrum ◽  
Dihydroxyacetone Phosphate ◽  
Enzyme Complex ◽  
Rabbit Muscle ◽  
Glycerophosphate Dehydrogenase ◽  
Dead End

Studies on initial velocity and product inhibition were carried out on crystalline cytoplasmic NAD+-linked L-α-glycerophosphate dehydrogenase from rabbit muscle, at pH 7.8 and 9.0 at 26 °C. Michaelis and inhibition constants for all the reactants were determined. The kinetic data were consistent with an ordered mechanism in which nicotinamide–adenine dinucleotide (NAD+) or its reduced form (NADH) is bound to the enzyme before the addition of the glycerophosphate (LαGP) or dihydroxyacetone phosphate (DHAP) respectively. At high concentrations NADH, DHAP, and LαGP, but not NAD+, produced substrate inhibition. Combined product-inhibition and dead-end inhibition studies indicated the formation of inactive dead-end complexes of NADH–enzyme, DHAP–enzyme, and LαGP–enzyme–NADH. The low rate constant calculated for the dissociation of the active NADH–enzyme complex suggested an ordered mechanism involving either the formation of an inactive dead-end NADH–enzyme complex or an isomerized NADH–enzyme complex. A choice between these possibilities could not be made on the basis of the present kinetic data. A mechanism for substrate inhibition involving two NAD+-binding sites per mole of enzyme is proposed. Alterations of the ultraviolet absorption spectrum of the enzyme by NAD+ and NADH were in agreement with the conclusion from the kinetic results that the coenzymes are bound to the enzyme before the substrates. DHAP and LαGP caused no alteration in the enzyme spectrum. Spectral changes compatible with the formation of ternary and dead-end complexes were also detected.

scholarly journals A study of the kinetics and mechanism of rabbit muscle l-glycerol 3-phosphate dehydrogenase

1974 ◽  
Vol 143 (1) ◽  
pp. 19-27 ◽  
Author(s):  
Philip Bentley ◽  
F. Mark Dickinson
Keyword(s):  
Basic Mechanism ◽  
Kinetics And Mechanism ◽  
Dihydroxyacetone Phosphate ◽  
Rabbit Muscle ◽  
Kinetics Of Oxidation ◽  
Mechanism Of Catalysis ◽  
Glycerol 3 Phosphate Dehydrogenase ◽  
Ph Range ◽  
Kinetics Of

1. The kinetics of oxidation of l-glycerol 3-phosphate by NAD+and of reduction of dihydroxyacetone phosphate by NADH catalysed by rabbit muscle glycerol 3-phosphate dehydrogenase were studied over the range pH6–9. 2. The enzyme was found to catalyse the oxidation of glyoxylate by NAD+at pH8.0 and the kinetics of this reaction were also studied. 3. The results are consistent with a compulsory mechanism of catalysis for glycerol 3-phosphate oxidation and dihydroxyacetone phosphate reduction in the intermediate regions of pH, but modifications to the basic mechanism are required to fully explain results at the extremes of the pH range, with these substrates and for glyoxylate oxidation at pH8.0.

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