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.

The role of secondary alcoholic groups of D-galacturonan in its degradation with exo-D-galacturonanase

1980 ◽  
Vol 45 (2) ◽  
pp. 427-434 ◽  
Author(s):  
Kveta Heinrichová ◽  
Rudolf Kohn
Keyword(s):  
Acetyl Derivative ◽  
Competitive Inhibitor ◽  
Hydroxyl Groups ◽  
Pectic Acid ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Individual ◽  
Degradation Degree ◽  
Derivatives Of

The effect of exo-D-galacturonanase from carrot on O-acetyl derivatives of pectic acid of variousacetylation degree was studied. Substitution of hydroxyl groups at C(2) and C(3) of D-galactopyranuronic acid units influences the initial rate of degradation, degree of degradation and its maximum rate, the differences being found also in the time of limit degradations of the individual O-acetyl derivatives. Value of the apparent Michaelis constant increases with increase of substitution and value of Vmax changes. O-Acetyl derivatives act as a competitive inhibitor of degradation of D-galacturonan. The extent of the inhibition effect depends on the degree of substitution. The only product of enzymic reaction is D-galactopyranuronic acid, what indicates that no degradation of the terminal substituted unit of O-acetyl derivative of pectic acid takes place. Substitution of hydroxyl groups influences the affinity of the enzyme towards the modified substrate. The results let us presume that hydroxyl groups at C(2) and C(3) of galacturonic unit of pectic acid are essential for formation of the enzyme-substrate complex.

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.

A KINETIC STUDY OF THE DEAMINATION OF SOME ADENOSINE ANALOGUES: SUBSTRATE SPECIFICITY OF ADENOSINE DEAMINASE

1966 ◽  
Vol 44 (3) ◽  
pp. 331-337 ◽  
Author(s):  
J. Lyndal York ◽  
G. A. LePage
Keyword(s):  
Substrate Specificity ◽  
Kinetic Study ◽  
Adenosine Deaminase ◽  
Competitive Inhibitor ◽  
Kinetic Constants ◽  
Glycosidic Linkage ◽  
Adenosine Analogues ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Marked Dependence

The kinetic constants Km and Vmax were determined for the deamination by adenosine deaminase of a series of analogues of adenosine containing "fraudulent" sugars. The configuration of the 2′-hydroxyl was found to be important for the binding of enzyme and substrate. The largest effect of changes in sugar structure was on the rate of breakdown of the enzyme–substrate complex to form products, i.e. Vmax. The nature of the configuration in the 3′-position was not important if the 2′-hydroxyl was trans to the glycosidic linkage; however, if the steric arrangement of the 2′-hydroxyl was cis to the glycosidic linkage, then Vmax showed a marked dependence on the nature of the 3′-substituent and its configuration. For instance, Vmax values were for arabinosyl adenine < 3′-deoxyarabinosyl adenine <lyxosyl adenine. 6-N-methyladenosine was found to be a competitive inhibitor of adenosine deaminase, with a Ki of 2 × 10−6M.

THE COMPETITIVE INHIBITION OF THE CYTOPLASMICL-α-GLYCEROPHOSPHATE DEHYDROGENASE OF SKELETAL MUSCLE BYL-α-GLYCEROPHOSPHATE

1965 ◽  
Vol 43 (1) ◽  
pp. 17-24 ◽  
Author(s):  
M. C. Blanchaer
Keyword(s):  
Skeletal Muscle ◽  
Phosphate Buffer ◽  
Competitive Inhibition ◽  
Mitochondrial Respiratory Chain ◽  
Competitive Inhibitor ◽  
Test System ◽  
Dihydroxyacetone Phosphate ◽  
Rabbit Muscle ◽  
Michaelis Constant ◽  
Glycerophosphate Dehydrogenase

The inhibition by L-α-glycerophosphate of the reduction of dihydroxyacetone phosphate by crystalline rabbit muscle NAD+-linked L-α-glycerophosphate dehydrogenase has been examined. As a result of the measurement of the absorbance at 340 mμ in a photometric test system at 26° containing 0.08–2.0 mM dihydroxyacetone phosphate, 0.14 mM NADH, and 1–1.5 μg crystalline enzyme in 1.5 ml 10 mM EDTA −0.1 M phosphate buffer at pH 7-0, the apparent Michaelis constant (Km) for dihydroxyacetone phosphate was found to be 0.363 mM (± 0.025 S.E.). L-α-Giycerophosphate, but not D-α-glycerophosphate, acted as a competitive inhibitor in this system with an apparent inhibition constant (Ki) of 0.575 mM (± 0.030). Substitution of 50 mM triethanolarnine buffer for the 0.1 M phosphate buffer lowered the Kmto 0.088 mM (± 0.019) and the Kito 0.240 mM (± 0.013). To study the enzyme at lower NADH concentrations, a fluorometric system containing 20–75 μM NADH, 5–370 μM DHAP, and 0.5–2.0 μg enzyme in 1 ml 2 mM EDTA −50 mM triethanolarnine buffer, pH 7.0 at 23°, was used. The apparent Kmfor dihydroxyacetone phosphate and Kifor L-α-glycerophosphate were 0.075 μM (± 0.020) and 0.186 mM (± 0.006) respectively, at a NADH concentration of 75 μM. Lowering the NADH concentration to 20 μM further decreased the apparent Kmand Kivalues to 0.039 mM (± 0.008) and 0.056 mM (± 0.007) respectively.A consideration of the concentrations of dihydroxyacetone phosphate and L-α-glycerophosphate in muscle during contraction suggests that the competitive inhibition of cytoplasmic L-α-glycerophosphate dehydrogenase by its product, L-α-glycerophosphate, may influence the pathway of triose phosphate utilization and also the coupling, by way of the L-α-glycerophosphate cycle, of cytoplasmic NADH-generating reactions to the mitochondrial respiratory chain.

scholarly journals Purification and properties of erythro-β-hydroxyasparate dehydratase from Micrococcus denitrificans

1965 ◽  
Vol 97 (2) ◽  
pp. 547-554 ◽  
Author(s):  
RG Gibbs ◽  
JG Morris
Keyword(s):  
Pyridoxal Phosphate ◽  
Competitive Inhibitor ◽  
The Novel ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Bivalent Cations ◽  
Purification And Properties ◽  
Key Enzyme ◽  
Aldolase Activity ◽  
No Activation

1. The novel enzyme, erythro-beta-hydroxyaspartate dehydratase, a key enzyme of the beta-hydroxyaspartate pathway (Kornberg & Morris, 1963, 1965), has been purified 30-fold from extracts of glycollate-grown Micrococcus denitrificans. The purified preparation was devoid of erythro-beta-hydroxyaspartate-aldolase activity, and free from enzymes that act on oxaloacetate. 2. Properties of the purified dehydratase were studied by direct assay of the enzymic formation of oxaloacetate and ammonia from added erythro-beta-hydroxyaspartate. 3. The enzyme was highly substrate-specific, utilizing only the l-isomer of erythro-beta-hydroxyaspartate (K(m), 0.43mm, and V(max.), 99mumoles of oxaloacetate formed/min./mg. of protein at pH9.15 and 30 degrees). Of many compounds tested, only maleate was a competitive inhibitor (K(i), 32mm at pH7.6). 4. The optimum pH for activity was about 9.5. The K(m) varied with pH, showing a marked optimum at pH7.8. The V(max.) also varied with pH in a manner suggesting the presence in the enzyme-substrate complex of a dissociable group of pK‣(a) about 8.5. 5. Carbonyl reagents were inhibitory, but of three thiol reagents tested only p-chloromercuribenzoate was inhibitory. 6. A partially resolved preparation of the enzyme was activated four-fold by the addition of pyridoxal phosphate and thereby restored to half activity. 7. EDTA (0.1mm) was almost completely inhibitory, activity being restored by bivalent cations (Mg(2+), Ca(2+) and Mn(2+)); no activation by univalent cations was observed. 8. The findings are discussed in the light of reported properties of related hydroxyamino acid dehydratases.

scholarly journals The Experimentally Determined Velocity of Catalysis could be Higher in the Absence of Sequestration

2019 ◽  
pp. 1-12
Author(s):  
Ikechukwu I. Udema ◽  
Abraham Olalere Onigbinde
Keyword(s):  
Maximum Velocity ◽  
Order Rate Constant ◽  
Free Enzyme ◽  
Theoretical Research ◽  
Coefficient Of Determination ◽  
First Order ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Pseudo First Order ◽  
The Relationship

Background: It is not unusual to observe calculated “total” free enzyme ([E]) in enzyme catalysed reaction, but this should include total enzyme-substrate complex ([EST]) which accounts for sequestration. Objectives: 1) To show indirectly that the velocities of catalytic action can be higher than experimentally observed velocities without sequestration and 2) redefine the relationship between velocity of hydrolysis with Michaelian enzyme and [E], where concentration of substrate, [ST] <  Michaelis-Menten constant, KM. Methods: A theoretical research and experimentation using Bernfeld method to determine velocities of amylolysis with which to mathematically calculate [EST] and the enzyme-substrate complex ([ES]) prepared for product, P, formation. Results: The [EST] is < [E]; [EST] and pseudo-first order constant, k decreased with increasing [ST] and increased with increasing concentration of enzyme [ET] while velocity amylolysis, v and maximum velocity of amylolysis, vmax expectedly increased with increasing [ET] and [ST]. Conclusion: The fact is that the [EST] is lower than what is usually referred to as free enzyme ([ET] - [ES]). Therefore, if the additional part of [EST] dissociated into product within the duration of assay, the velocity of amylolysis could be higher. The most important outcome and corollary when [KM] > [ST] is that v a 1/[E], v a [E][ST] and a quadratic relationship exists between pseudo-first order rate constant and maximum velocity of amylolysis; separately, v is not a [E] and if v a [ST] (if v/[ST] is constant with coefficient of determination = 1), then KM is not applicable.

scholarly journals Photoenzymatic Repair of Ultraviolet Damage in DNA

1962 ◽  
Vol 45 (4) ◽  
pp. 725-741 ◽  
Author(s):  
Claud S. Rupert
Keyword(s):  
Heavy Metals ◽  
Quantum Yield ◽  
Kinetic Studies ◽  
Order Rate Constant ◽  
Reaction Scheme ◽  
First Order ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Exponential Decrease ◽  
Ultraviolet Damage

The photoenzyme from bakers' yeast which repairs ultraviolet-inactivated transforming DNA is mechanically bound to ultraviolet-irradiated DNA in the dark, but not to unirradiated DNA. In the bound condition it is stabilized against inactivation by heat and heavy metals. Both the mechanical binding and stabilization are eliminated by illumination. These observations are consistent with the reaction scheme suggested by kinetic studies, in which the enzyme combines with the ultraviolet lesions in DNA and the complex absorbs light, producing repair and subsequent liberation of the enzyme. The approximately exponential decrease of heat stabilization during illumination gives the first order rate constant for the light-dependent step at the corresponding light intensity. This quantity in turn sets limits on the possible magnitude of the molar absorption coefficient of the enzyme-substrate complex and on the quantum yield of the process.

Transient Formation of a Complex Between α-Chymotrypsin, Proflavin, and Tosylarginine Methyl Ester (TAME)

1972 ◽  
Vol 50 (3) ◽  
pp. 257-260 ◽  
Author(s):  
George H. Czerlinski ◽  
Catherine Odell
Keyword(s):  
Methyl Ester ◽  
Ternary Complex ◽  
Competitive Inhibitor ◽  
Relaxation Processes ◽  
Relaxation Time Constant ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Fast Relaxation ◽  
Chemical Relaxation ◽  
Relaxation Experiments

Chemical relaxation experiments were conducted on the reaction of α-chymotrypsin, with the competitive inhibitor proflavin and the substrate analogue TAME (tosylarginine methyl ester) in phosphate buffer, pH 6.7, observing transmission changes at 465 mμ. Two chemical relaxation processes were observed with the slow one attributed to a monomolecular interconversion of the enzyme–substrate complex. The concentration dependence of the reciprocal fast relaxation time constant only agrees with the equations derived for the involvement of a labile ternary complex between enzyme, substrate, and inhibitor (as simplest model).

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