scholarly journals pH and kinetic studies of chloroplast sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea)

1988 ◽  
Vol 253 (1) ◽  
pp. 249-254 ◽  
Author(s):  
F Cadet ◽  
J C Meunier
Keyword(s):  
Spinacia Oleracea ◽  
Ph Dependence ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Kinetic Properties ◽  
Enzyme Specificity ◽  
Active Centre ◽  
Specificity Constant ◽  
The One ◽  
Fructose 1,6 Bisphosphate

The aim of this paper is to study some steady-state kinetic properties of sedoheptulose-1,7-bisphosphatase, its pH-dependence and the effect of a substrate analogue, fructose 2,6-bisphosphate. Studies were carried out with sedoheptulose 1,7-bisphosphate and with fructose 1,6-bisphosphate, an alternative substrate. The pK values are identical for both substrates, and fructose 2,6-bisphosphate behaves like a competitive inhibitor. These results suggest that there exists a unique active site for either sedoheptulose 1,7-bisphosphate or fructose 1,6-bisphosphate on the enzyme molecule. Increasing Mg2+ concentrations shifted the optimum pH. As for fructose-1,6-bisphosphatase, we believe that this shift is due to the neutralization of negative charges near the active centre [Cadet, Meunier & Ferté (1987) Eur. J. Biochem. 162, 393-398]. The free species of sedoheptulose 1,7-bisphosphate and fructose 1,6-bisphosphate are not the usual substrates of enzyme, nor is Mg2+. But the kinetics relative to the (Mg2+-substrate4-)2- complex is not consistent with this complex being the substrate. An explanation of this discrepancy is proposed, involving both the negative charges near the active centre and the positive charges of Mg2+. The observed Vmax. of the reduced enzyme is 65% of the theoretical Vmax. for both substrates, but the observed Vmax. relative to sedoheptulose 1,7-bisphosphate is 3 times the one relative to fructose 1,6-bisphosphate. The specificity constant (kcat./Km), 1.62 × 10(6) M-1.s-1 with respect to sedoheptulose 1,7-bisphosphate compared with 5.5 × 10(4) M-1.s-1 with respect to fructose 1,6-bisphosphate, indicates that the enzyme specificity towards sedoheptulose 1,7-bisphosphate is high but not absolute.

scholarly journals Properties of oxidized and reduced spinach (Spinacia oleracea) chloroplast fructose-1,6-bisphosphatase activated by various agents

1991 ◽  
Vol 278 (3) ◽  
pp. 787-791 ◽  
Author(s):  
T Chardot ◽  
J C Meunier
Keyword(s):  
Spinacia Oleracea ◽  
Acid Group ◽  
Lag Phase ◽  
Kinetic Properties ◽  
Amino Acid Group ◽  
Progress Curve ◽  
Fructose 1,6 Bisphosphatase ◽  
Specificity Constant ◽  
The Stability ◽  
Fructose 1,6 Bisphosphate

Fructose-1,6-bisphosphatase (FBPase) can be reduced and activated by either dithiothreitol or reduced thioredoxin. This activation is pH-dependent. An amino acid group with a pK value of 5.55 is involved in this process. Both enzyme forms can also be stimulated by agents such as fructose 1,6-bisphosphate, Mg2+, Ca2+ and Ca2+/fructose 1,6-bisphosphate. FBPase reduced by dithiothreitol is more strongly activated than the enzyme reduced by thioredoxin. The specificity constant (kcat./Km) is enhanced over 2.5-25-fold and 1.5-2-fold (depending on the agent used) for FBPase reduced by dithiothreitol and thioredoxin respectively. In both cases, no new kinetic properties appeared. The pH-activity profile of the stimulated enzyme is slightly shifted towards the acidic side with respect to the reduced enzyme. A lag phase is observed in the progress curve of both enzymic forms, treated or untreated. Each agent used to stimulate must induce a new conformation of the enzyme, more active than the initial one, characterized by a specificity constant and a relaxation time. This lag phase tends to disappear when the assay temperature is increased. Temperature has the same effect on the activity of oxidized, reduced and stimulated FBPase, but different effects on the stability of the different forms.

Kinetic Studies of Platelet Aggregation Induced by Adenosine Diphosphate and Its Inhibition by Chelating Agents, Guanidino Compounds, and Adenosine

1967 ◽  
Vol 18 (03/04) ◽  
pp. 713-725 ◽  
Author(s):  
L Skoza ◽  
Marjorie B Zucker ◽  
Z Jerushalmy ◽  
R Grant
Keyword(s):  
Platelet Aggregation ◽  
Ph Dependence ◽  
Kinetic Studies ◽  
Adenosine Diphosphate ◽  
Competitive Inhibitor ◽  
Ionized Calcium ◽  
Guanidino Compounds ◽  
Lag Period ◽  
A Chain ◽  
Citrate Dextrose

SummaryThe kinetics, activation, pH dependence and inhibition of platelet aggregation induced by ADP were studied by recording the OD of stirred PRP from blood anticoagulated with acid-citrate-dextrose solution. There was a lag period between the addition of ADP and the initiation of aggregation. Addition of ADP in the absence of stirring or ionized calcium did not cause aggregation. When aggregation was subsequently initiated by stirring or the addition of CaCl2 there was no lag period. The steepest slope of the OD curve was used as the reaction velocity (V). It was affected by the concentration of calcium ions and was maximum at pH 8.0. When the ADP concentration was varied in the presence of different concentrations of ionized calcium, the overall kinetics revealed a series of rectangular hyperbola segments which did not pass through the origin. These observations led to the conclusion that the overall reaction can be depicted as a chain reaction: ADP interacts reversibly with platelets; when calcium is present these platelets become “sticky” and, when stirred, they aggregate. Kinetic studies of inhibition indicated that adenosine is a competitive inhibitor of ADP. The guanidino compounds tested were noncompetitive with respect to ADP but their inhibitory activity was counteracted by calcium.

scholarly journals Kinetic properties and tissular distribution of mammalian phosphomannomutase isozymes

1999 ◽  
Vol 339 (1) ◽  
pp. 201-207 ◽  
Author(s):  
Michel PIRARD ◽  
Younes ACHOURI ◽  
Jean-François COLLET ◽  
Els SCHOLLEN ◽  
Gert MATTHIJS ◽  
...  
Keyword(s):  
Kinetic Studies ◽  
Time Dependent ◽  
Chemical Characteristics ◽  
Kinetic Properties ◽  
Rat Tissues ◽  
Northern Blots ◽  
Carbohydrate Deficient Glycoprotein Syndrome ◽  
Fructose 1,6 Bisphosphate ◽  
Acyl Phosphate ◽  
Stimulation Of

Human tissues contain two types of phosphomannomutase, PMM1 and PMM2. Mutations in the PMM2 gene are responsible for the most common form of carbohydrate-deficient glycoprotein syndrome [Matthijs, Schollen, Pardon, Veiga-da-Cunha, Jaeken, Cassiman and Van Schaftingen (1997) Nat. Genet.19, 88–92]. The protein encoded by this gene has now been produced in Escherichia coli and purified to homogeneity, and its properties have been compared with those of recombinant human PMM1. PMM2 converts mannose 1-phosphate into mannose 6-phosphate about 20 times more rapidly than glucose 1-phosphate to glucose 6-phosphate, whereas PMM1 displays identical Vmax values with both substrates. The Ka values for both mannose 1,6-bisphosphate and glucose 1,6-bisphosphate are significantly lower in the case of PMM2 than in the case of PMM1. Like PMM1, PMM2 forms a phosphoenzyme with the chemical characteristics of an acyl-phosphate. PMM1 and PMM2 hydrolyse different hexose bisphosphates (glucose 1,6-bisphosphate, mannose 1,6-bisphosphate, fructose 1,6-bisphosphate) at maximal rates of ≈ 3.5 and 0.3% of their PMM activity, respectively. Fructose 1,6-bisphosphate does not activate PMM2 but causes a time-dependent stimulation of PMM1 due to the progressive formation of mannose 1,6-bisphosphate from fructose 1,6-bisphosphate and mannose 1-phosphate. Experiments with specific antibodies, kinetic studies and Northern blots indicated that PMM2 is the only detectable isozyme in most rat tissues except brain and lung, where PMM1 accounts for about 66 and 13% of the total activities, respectively.

scholarly journals Covalent modification and site-directed mutagenesis of an active site tryptophan of human prostatic acid phosphatase.

1997 ◽  
Vol 44 (4) ◽  
pp. 659-672 ◽  
Author(s):  
Z Zhang ◽  
K Ostanin ◽  
R L Van Etten
Keyword(s):  
Acid Phosphatase ◽  
Active Site ◽  
Ph Dependence ◽  
Prostatic Acid Phosphatase ◽  
Competitive Inhibitor ◽  
Covalent Modification ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Phosphate Binding

Because tryptophans are found as part of the phosphate binding sites in a number of proteins, human prostatic acid phosphatase (hPAP) was examined for the presence and the role of essential tryptophan residues. The pH dependence of the intrinsic fluorescence of hPAP resembled the kinetic pH dependence. Chemical modification by N-bromosuccinimide (NBS) resulted in an inactivation of the enzyme and produced a characteristic reduction of the protein absorbance at 280 nm. Two tryptophans per subunit were modified, and this was accompanied by an apparently complete loss of enzymatic activity. In the presence of the competitive inhibitor L-(+)-tartrate, the loss of enzyme activity was significantly reduced as compared to the rate of tryptophan modification. After labeling the protein with 2,4-dinitrophenylsulfenyl chloride (DNPS-Cl), two tryptic peptides containing DNPS-labeled tryptophans were isolated and the sequences were identified by amino acid sequence analysis and mass spectroscopy. One peptide corresponded to residues 172-176, and included Trp174. The other corresponded to the C-terminal sequence, including Trp336. It was concluded that Trp174 was at the active site of the human enzyme because it was protected by the competitive inhibitor tartrate in the DNPS-Cl modification studies. This is also consistent with the location of a homologous residue in the structure of the rat enzyme. Using site-directed mutagenesis, Trp174 was replaced by Phe or Leu. Both mutants showed altered kinetic properties, including lower Km values with several aromatic substrates, and also exhibited reduced stability towards urea denaturation.

scholarly journals Universal Capability of 3-Ketosteroid Δ1-Dehydrogenases to Catalyze Δ1-Dehydrogenation of C17- Substituted Steroids

2021 ◽  
Author(s):  
Patrycja Wójcik ◽  
Michał Glanowski ◽  
Agnieszka M. Wojtkiewicz ◽  
Ali Rohman ◽  
M. Szaleniec
Keyword(s):  
Substrate Specificity ◽  
Rhodococcus Erythropolis ◽  
Kinetic Studies ◽  
Similar Rate ◽  
Enzyme Specificity ◽  
The Poor ◽  
Steady State Kinetics ◽  
Ping Pong ◽  
Specificity Constant ◽  
Dynamics Simulations

Abstract Background 3-Ketosteroid Δ1-dehydrogenases (KSTDs) are the enzymes involved in microbial cholesterol degradation and modification of steroids. They catalyze dehydrogenation between C1 and C2 atoms in ring A of the polycyclic structure of 3-ketosteroids. KSTDs substrate spectrum is broad, even though most of them prefer steroids with small substituents at the C17 atom. The investigation of the KSTD’s substrate specificity is hindered by the poor solubility of the hydrophobic steroids in aqueous solutions. In this paper, we used 2-hydroxpropyl-β-cyclodextrin (HBC) as a solubilizing agent in a study of the KSTDs steady-state kinetics and demonstrated that substrate bioavailability has a pivotal impact on enzyme specificity. Results Molecular dynamics simulations on KSTD1 from Rhodococcus erythropolis indicated no difference in ΔGbind between the native substrate, androst-4-en-3,17-dione (AD; − 8.02 kcal/mol), and more complex steroids such as cholest-4-en-3-one (–8.40 kcal/mol) or diosgenone (–6.17 kcal/mol). No structural obstacle for binding of the extended substrates was also observed. Following this observation, our kinetic studies conducted in the presence of HBC confirmed KSTD1 activity towards both types of steroids. We have compared the substrate specificity of KSTD1 to the other enzyme known for its activity with cholest-4-en-3-one, KSTD from Sterolibacterium denitrificans (AcmB). The addition of solubilizing agent caused AcmB to exhibit a higher affinity to cholest-4-en-3-one (Ping-Pong bi bi KmA= 23.7 µM) than to AD (KmA= 529.2 µM), a supposedly native substrate of the enzyme. Moreover, we have isolated AcmB isoenzyme (AcmB2) and showed that conversion of AD and cholest-4-en-3-one proceeds at a similar rate. We demonstrated also that the apparent specificity constant of AcmB for cholest-4-en-3-one (kcat/KmA= 9.25 ∙ 106 M− 1 s− 1) is almost 20 times higher than measured for KSTD1 (kcat/KmA= 4.71 ∙ 105 M− 1 s− 1). Conclusions We confirmed the existence of AcmB preference for a substrate with an undegraded isooctyl chain. However, we showed that KSTD1 which was reported to be inactive with such substrates can catalyze the reaction if the solubility problem is addressed.

scholarly journals Inhibition of monoamine oxidase by substituted hydrazines

1972 ◽  
Vol 128 (4) ◽  
pp. 913-919 ◽  
Author(s):  
Keith F. Tipton
Keyword(s):  
Monoamine Oxidase ◽  
Time Course ◽  
Ph Dependence ◽  
Protective Action ◽  
Competitive Inhibitor ◽  
Reversible Inhibitor ◽  
Kinetic Properties ◽  
Irreversible Inhibitor ◽  
Time Courses ◽  
Substituted Hydrazines

1. The initial rate of inhibition of monoamine oxidase by phenethylhydrazine was shown to be similar, in pH-dependence and kinetic properties, to the oxidation of that compound by monoamine oxidase. 2. The time-course of irreversible inhibition of monoamine oxidase by phenethylhydrazine lags behind that of reversible inhibition. 3. Hydralzine was shown to be a reversible competitive inhibitor of monoamine oxidase, but phenylhydrazine is an irreversible inhibitor. Inhibition by the latter compound is not affected by the absence of oxygen, and the presence of substrate exerts no protective action. 4. Hydrazine does not inhibit monoamine oxidase unless a substrate and oxygen are present. 5. Phenethylidenehydrazine was found to be a time-dependent inhibitor of monoamine oxidase and the rate of inhibition was hindered by increasing oxygen concentration. 6. A mechanism for the inhibition of the enzyme by phenethylhydrazine is proposed in which the product of oxidation of this compound is a potent reversible inhibitor and an irreversible inhibitor of the enzyme. A computer simulation of such a mechanism predicts time-courses of inhibition that are in reasonable agreement with those observed experimentally.

scholarly journals Universal capability of 3-ketosteroid Δ1-dehydrogenases to catalyze Δ1-dehydrogenation of C17-substituted steroids

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Patrycja Wójcik ◽  
Michał Glanowski ◽  
Agnieszka M. Wojtkiewicz ◽  
Ali Rohman ◽  
Maciej Szaleniec
Keyword(s):  
Substrate Specificity ◽  
Rhodococcus Erythropolis ◽  
Kinetic Studies ◽  
Similar Rate ◽  
Enzyme Specificity ◽  
The Poor ◽  
Steady State Kinetics ◽  
Ping Pong ◽  
Specificity Constant ◽  
Dynamics Simulations

Abstract Background 3-Ketosteroid Δ1-dehydrogenases (KSTDs) are the enzymes involved in microbial cholesterol degradation and modification of steroids. They catalyze dehydrogenation between C1 and C2 atoms in ring A of the polycyclic structure of 3-ketosteroids. KSTDs substrate spectrum is broad, even though most of them prefer steroids with small substituents at the C17 atom. The investigation of the KSTD’s substrate specificity is hindered by the poor solubility of the hydrophobic steroids in aqueous solutions. In this paper, we used 2-hydroxpropyl-β-cyclodextrin (HBC) as a solubilizing agent in a study of the KSTDs steady-state kinetics and demonstrated that substrate bioavailability has a pivotal impact on enzyme specificity. Results Molecular dynamics simulations on KSTD1 from Rhodococcus erythropolis indicated no difference in ΔGbind between the native substrate, androst-4-en-3,17-dione (AD; − 8.02 kcal/mol), and more complex steroids such as cholest-4-en-3-one (− 8.40 kcal/mol) or diosgenone (− 6.17 kcal/mol). No structural obstacle for binding of the extended substrates was also observed. Following this observation, our kinetic studies conducted in the presence of HBC confirmed KSTD1 activity towards both types of steroids. We have compared the substrate specificity of KSTD1 to the other enzyme known for its activity with cholest-4-en-3-one, KSTD from Sterolibacterium denitrificans (AcmB). The addition of solubilizing agent caused AcmB to exhibit a higher affinity to cholest-4-en-3-one (Ping-Pong bi bi KmA = 23.7 μM) than to AD (KmA = 529.2 μM), a supposedly native substrate of the enzyme. Moreover, we have isolated AcmB isoenzyme (AcmB2) and showed that conversion of AD and cholest-4-en-3-one proceeds at a similar rate. We demonstrated also that the apparent specificity constant of AcmB for cholest-4-en-3-one (kcat/KmA = 9.25∙106 M−1 s−1) is almost 20 times higher than measured for KSTD1 (kcat/KmA = 4.71∙105 M−1 s−1). Conclusions We confirmed the existence of AcmB preference for a substrate with an undegraded isooctyl chain. However, we showed that KSTD1 which was reported to be inactive with such substrates can catalyze the reaction if the solubility problem is addressed.

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.

α-Retaining glucosyl transfer catalysed by trehalose phosphorylase from Schizophyllum commune: mechanistic evidence obtained from steady-state kinetic studies with substrate analogues and inhibitors

2001 ◽  
Vol 360 (3) ◽  
pp. 727-736 ◽  
Author(s):  
Bernd NIDETZKY ◽  
Christian EIS
Keyword(s):  
Steady State ◽  
Transition State ◽  
Schizophyllum Commune ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Chemical Mechanism ◽  
Steady State Kinetic ◽  
State Kinetic ◽  
Trehalose Phosphorylase

Fungal trehalose phosphorylase is classified as a family 4 glucosyltransferase that catalyses the reversible phosphorolysis of α,α-trehalose with net retention of anomeric configuration. Glucosyl transfer to and from phosphate takes place by the partly rate-limiting interconversion of ternary enzyme–substrate complexes formed from binary enzyme–phosphate and enzyme–α-d-glucopyranosyl phosphate adducts respectively. To advance a model of the chemical mechanism of trehalose phosphorylase, we performed a steady-state kinetic study with the purified enzyme from the basidiomycete fungus Schizophyllum commune by using alternative substrates, inhibitors and combinations thereof in pairs as specific probes of substrate-binding recognition and transition-state structure. Orthovanadate is a competitive inhibitor against phosphate and α-d-glucopyranosyl phosphate, and binds 3×104-fold tighter (Ki≈ 1μM) than phosphate. Structural alterations of d-glucose at C-2 and O-5 are tolerated by the enzyme at subsite +1. They lead to parallel effects of approximately the same magnitude (slope = 1.14; r2 = 0.98) on the reciprocal catalytic efficiency for reverse glucosyl transfer [log (Km/kcat)] and the apparent affinity of orthovanadate determined in the presence of the respective glucosyl acceptor (log Ki). An adduct of orthovanadate and the nucleophile/leaving group bound at subsite +1 is therefore the true inhibitor and displays partial transition state analogy. Isofagomine binds to subsite −1 in the enzyme–phosphate complex with a dissociation constant of 56μM and inhibits trehalose phosphorylase at least 20-fold better than 1-deoxynojirimycin. The specificity of the reversible azasugars inhibitors would be explained if a positive charge developed on C-1 rather than O-5 in the proposed glucosyl cation-like transition state of the reaction. The results are discussed in the context of α-retaining glucosyltransferase mechanisms that occur with and without a β-glucosyl enzyme intermediate.

scholarly journals Signaling pathways have an inherent need for noise to acquire information

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Eugenio Azpeitia ◽  
Eugenio P. Balanzario ◽  
Andreas Wagner
Keyword(s):  
Signaling Pathways ◽  
Information Acquisition ◽  
Cellular Level ◽  
Kinetic Properties ◽  
Positive Role ◽  
Binding Interactions ◽  
Reversible Binding ◽  
Wide Range ◽  
The One ◽  
Parameter Values

Abstract Background All living systems acquire information about their environment. At the cellular level, they do so through signaling pathways. Such pathways rely on reversible binding interactions between molecules that detect and transmit the presence of an extracellular cue or signal to the cell’s interior. These interactions are inherently stochastic and thus noisy. On the one hand, noise can cause a signaling pathway to produce the same response for different stimuli, which reduces the amount of information a pathway acquires. On the other hand, in processes such as stochastic resonance, noise can improve the detection of weak stimuli and thus the acquisition of information. It is not clear whether the kinetic parameters that determine a pathway’s operation cause noise to reduce or increase the acquisition of information. Results We analyze how the kinetic properties of the reversible binding interactions used by signaling pathways affect the relationship between noise, the response to a signal, and information acquisition. Our results show that, under a wide range of biologically sensible parameter values, a noisy dynamic of reversible binding interactions is necessary to produce distinct responses to different stimuli. As a consequence, noise is indispensable for the acquisition of information in signaling pathways. Conclusions Our observations go beyond previous work by showing that noise plays a positive role in signaling pathways, demonstrating that noise is essential when such pathways acquire information.

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