scholarly journals Re-examination of the roles of PEP and Mg2+ in the reaction catalysed by the phosphorylated and non-phosphorylated forms of phosphoenolpyruvate carboxylase from leaves of Zea mays

1998 ◽  
Vol 332 (3) ◽  
pp. 633-642 ◽  
Author(s):  
Alejandro TOVAR-MÉNDEZ ◽  
Rogelio RODRÍGUEZ-SOTRES ◽  
Dulce M. LÓPEZ-VALENTÍN ◽  
Rosario A. MUÑOZ-CLARES
Keyword(s):  
Active Site ◽  
Phosphoenolpyruvate Carboxylase ◽  
Metal Ion ◽  
Dissociation Constants ◽  
Physiological Role ◽  
Free Enzyme ◽  
Kinetic Properties ◽  
Allosteric Site ◽  
Physiological Concentration

To study the effects of phosphoenolpyruvate (PEP) and Mg2+ on the activity of the non-phosphorylated and phosphorylated forms of phosphoenolpyruvate carboxylase (PEPC) from Zea maysleaves, steady-state measurements have been carried out with the free forms of PEP (fPEP) and Mg2+ (fMg2+), both in a near-physiological concentration range. At pH 7.3, in the absence of activators, the initial velocity data obtained with both forms of the enzyme are consistent with the exclusive binding of MgPEP to the active site and of fPEP to an activating allosteric site. At pH 8.3, and in the presence of saturating concentrations of glucose 6-phosphate (Glc6P) or Gly, the free species also combined with the active site in the free enzyme, but with dissociation constants at least 35-fold that estimated for MgPEP. The latter dissociation constant was lowered to the same extent by saturating Glc6P and Gly, to approx. one-tenth and one-sixteenth in the non-phosphorylated and phosphorylated enzymes respectively. When Glc6P is present, fPEP binds to the active site in the free enzyme better than fMg2+, whereas the metal ion binds better in the presence of Gly. Saturation of the enzyme with Glc6P abolished the activation by fPEP, consistent with a common binding site, whereas saturation with Gly increased the affinity of the allosteric site for fPEP. Under all the conditions tested, our results suggest that fPEP is not able to combine with the allosteric site in the free enzyme, i.e. it cannot combine until after MgPEP, fPEP or fMg2+ are bound at the active site. The physiological role of Mg2+ in the regulation of the enzyme is only that of a substrate, mainly as part of the MgPEP complex. The kinetic properties of maize leaf PEPC reported here are consistent with the enzyme being well below saturation under the physiological concentrations of fMg2+ and PEP, particularly during the dark period; it is therefore suggested that the basal PEPC activity in vivois very low, but highly responsive to even small changes in the intracellular concentration of its substrate and effectors.

scholarly journals Structural and biochemical evidence of the glucose 6-phosphate-allosteric site of maize C4-phosphoenolpyruvate carboxylase: its importance in the overall enzyme kinetics

2020 ◽  
Vol 477 (11) ◽  
pp. 2095-2114
Author(s):  
Rosario A. Muñoz-Clares ◽  
Lilian González-Segura ◽  
Javier Andrés Juárez-Díaz ◽  
Carlos Mújica-Jiménez
Keyword(s):  
Active Site ◽  
Phosphoenolpyruvate Carboxylase ◽  
Site Directed Mutagenesis ◽  
Allosteric Site ◽  
Allosteric Activation ◽  
Physiological Relevance ◽  
Phosphorylated Compounds ◽  
In The Wild ◽  
Kinetics Of

Activation of phosphoenolpyruvate carboxylase (PEPC) enzymes by glucose 6-phosphate (G6P) and other phospho-sugars is of major physiological relevance. Previous kinetic, site-directed mutagenesis and crystallographic results are consistent with allosteric activation, but the existence of a G6P-allosteric site was questioned and competitive activation—in which G6P would bind to the active site eliciting the same positive homotropic effect as the substrate phosphoenolpyruvate (PEP)—was proposed. Here, we report the crystal structure of the PEPC-C4 isozyme from Zea mays with G6P well bound into the previously proposed allosteric site, unambiguously confirming its existence. To test its functionality, Asp239—which participates in a web of interactions of the protein with G6P—was changed to alanine. The D239A variant was not activated by G6P but, on the contrary, inhibited. Inhibition was also observed in the wild-type enzyme at concentrations of G6P higher than those producing activation, and probably arises from G6P binding to the active site in competition with PEP. The lower activity and cooperativity for the substrate PEP, lower activation by glycine and diminished response to malate of the D239A variant suggest that the heterotropic allosteric activation effects of free-PEP are also abolished in this variant. Together, our findings are consistent with both the existence of the G6P-allosteric site and its essentiality for the activation of PEPC enzymes by phosphorylated compounds. Furthermore, our findings suggest a central role of the G6P-allosteric site in the overall kinetics of these enzymes even in the absence of G6P or other phospho-sugars, because of its involvement in activation by free-PEP.

scholarly journals Kinetic properties and physiological role of the plastoquinone terminal oxidase (PTOX) in a vascular plant

2012 ◽  
Vol 1817 (12) ◽  
pp. 2140-2148 ◽  
Author(s):  
Martin Trouillard ◽  
Maryam Shahbazi ◽  
Lucas Moyet ◽  
Fabrice Rappaport ◽  
Pierre Joliot ◽  
...  
Keyword(s):  
Vascular Plant ◽  
Physiological Role ◽  
Kinetic Properties ◽  
Terminal Oxidase

Distinct functional roles of the two terminal halves of eukaryotic phosphofructokinase

2012 ◽  
Vol 445 (2) ◽  
pp. 213-218 ◽  
Author(s):  
Oscar H. Martínez-Costa ◽  
Valentina Sánchez ◽  
Antonio Lázaro ◽  
Eloy D. Hernández ◽  
Keith Tornheim ◽  
...  
Keyword(s):  
Active Site ◽  
Kinetic Properties ◽  
Wild Type ◽  
Allosteric Site ◽  
Functional Roles ◽  
Allosteric Interactions ◽  
C Terminus ◽  
N Terminus ◽  
Metabolite Binding

Eukaryotic PFK (phosphofructokinase), a key regulatory enzyme in glycolysis, has homologous N- and C-terminal domains thought to result from duplication, fusion and divergence of an ancestral prokaryotic gene. It has been suggested that both the active site and the Fru-2,6-P2 (fructose 2,6-bisphosphate) allosteric site are formed by opposing N- and C-termini of subunits orientated antiparallel in a dimer. In contrast, we show in the present study that in fact the N-terminal halves form the active site, since expression of the N-terminal half of the enzymes from Dictyostelium discoideum and human muscle in PFK-deficient yeast restored growth on glucose. However, the N-terminus alone was not stable in vitro. The C-terminus is not catalytic, but is needed for stability of the enzyme, as is the connecting peptide that normally joins the two domains (here included in the N-terminus). Co-expression of homologous, but not heterologous, N- and C-termini yielded stable fully active enzymes in vitro with sizes and kinetic properties similar to those of the wild-type tetrameric enzymes. This indicates that the separately translated domains can fold sufficiently well to bind to each other, that such binding of complementary domains is stable and that the alignment is sufficiently accurate and tight as to preserve metabolite binding sites and allosteric interactions.

The isolation, properties, and physiological role of lactic dehydrogenase from soybean cotyledons

1970 ◽  
Vol 48 (3) ◽  
pp. 533-540 ◽  
Author(s):  
J. King
Keyword(s):  
Specific Activity ◽  
Sulfhydryl Group ◽  
Physiological Role ◽  
Lactic Dehydrogenase ◽  
Reverse Direction ◽  
Kinetic Properties ◽  
Soybean Seeds ◽  
Time Period ◽  
Glycine Max L

L-Lactate:NAD oxidoreductase (EC. 1.1.1.27) was purified 110-fold from non-green soybean (Glycine max L. var. Canadian No. 1) cotyledons, and some of its kinetic properties were studied and compared to the properties of lactic dehydrogenases isolated from animals and microorganisms. The soybean enzyme was specific for L-lactate and NAD+ but in the reverse direction reduced not only pyruvate but also hydroxypyruvate and glyoxylate in the presence of NADH, although pyruvate was shown to be the preferred substrate. Optimum activity occurred at pH 9.2 in the direction of pyruvate formation and at pH 7.0 in the reverse direction. In its response to the use of coenzyme analogues and to heat treatment it resembled closely the L-lactic dehydrogenase from Lactobacillus plantarum. Its responses to acrylamide gel electrophoresis and to sulfhydryl group inhibitors were comparable to those of similar enzymes from animal sources.The physiological role of the enzyme in germinating soybean seeds, especially during the first 30 h when anaerobic conditions obtain within the seed, was assessed by measuring its specific activity and also by measuring the rise and fall of lactic acid concentration in cotyledons over the same time period. Various aspects of the metabolism of germinating fatty seeds are discussed in relation to this and other work recently reported.

scholarly journals Distribution of exogenous [125I]-3-iodothyronamine in mouse in vivo: relationship with trace amine-associated receptors

2012 ◽  
Vol 213 (3) ◽  
pp. 223-230 ◽  
Author(s):  
Grazia Chiellini ◽  
Paola Erba ◽  
Vittoria Carnicelli ◽  
Chiara Manfredi ◽  
Sabina Frascarelli ◽  
...  
Keyword(s):  
Kidney Tissue ◽  
Physiological Role ◽  
Physiological Concentration ◽  
Systemic Distribution ◽  
Trace Amine ◽  
Liver And Kidney ◽  
Residual Radioactivity ◽  
G Protein Coupled

3-Iodothyronamine (T1AM) is a novel chemical messenger, structurally related to thyroid hormone, able to interact with G protein-coupled receptors known as trace amine-associated receptors (TAARs). Little is known about the physiological role of T1AM. In this prospective, we synthesized [125I]-T1AM and explored its distribution in mouse after injecting in the tail vein at a physiological concentration (0.3 nM). The expression of the nine TAAR subtypes was evaluated by quantitative real-time PCR. [125I]-T1AM was taken up by each organ. A significant increase in tissue vs blood concentration occurred in gallbladder, stomach, intestine, liver, and kidney. Tissue radioactivity decreased exponentially over time, consistent with biliary and urinary excretion, and after 24 h, 75% of the residual radioactivity was detected in liver, muscle, and adipose tissue. TAARs were expressed only at trace amounts in most of the tissues, the exceptions being TAAR1 in stomach and testis and TAAR8 in intestine, spleen, and testis. Thus, while T1AM has a systemic distribution, TAARs are only expressed in certain tissues suggesting that other high-affinity molecular targets besides TAARs exist.

scholarly journals Evolutionary Expansion of the Amidohydrolase Superfamily in Bacteria in Response to the Synthetic Compounds Molinate and Diuron

2015 ◽  
Vol 81 (7) ◽  
pp. 2612-2624 ◽  
Author(s):  
Elena Sugrue ◽  
Nicholas J. Fraser ◽  
Davis H. Hopkins ◽  
Paul D. Carr ◽  
Jeevan L. Khurana ◽  
...  
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Active Sites ◽  
Evolutionary Transition ◽  
Functional Changes ◽  
Metal Ion Analysis ◽  
Amidohydrolase Superfamily ◽  
Kinetics Of ◽  
Hydrolysis Of

ABSTRACTThe amidohydrolase superfamily has remarkable functional diversity, with considerable structural and functional annotation of known sequences. In microbes, the recent evolution of several members of this family to catalyze the breakdown of environmental xenobiotics is not well understood. An evolutionary transition from binuclear to mononuclear metal ion coordination at the active sites of these enzymes could produce large functional changes such as those observed in nature, but there are few clear examples available to support this hypothesis. To investigate the role of binuclear-mononuclear active-site transitions in the evolution of new function in this superfamily, we have characterized two recently evolved enzymes that catalyze the hydrolysis of the synthetic herbicides molinate (MolA) and phenylurea (PuhB). In this work, the crystal structures, mutagenesis, metal ion analysis, and enzyme kinetics of both MolA and PuhB establish that these enzymes utilize a mononuclear active site. However, bioinformatics and structural comparisons reveal that the closest putative ancestor of these enzymes had a binuclear active site, indicating that a binuclear-mononuclear transition has occurred. These proteins may represent examples of evolution modifying the characteristics of existing catalysts to satisfy new requirements, specifically, metal ion rearrangement leading to large leaps in activity that would not otherwise be possible.

scholarly journals Calcium-binding properties of human erythrocyte calpain

1997 ◽  
Vol 325 (3) ◽  
pp. 721-726 ◽  
Author(s):  
Mauro MICHETTI ◽  
Franca SALAMINO ◽  
Roberto MINAFRA ◽  
Edon MELLONI ◽  
Sandro PONTREMOLI
Keyword(s):  
Active Site ◽  
Human Erythrocyte ◽  
Binding Sites ◽  
Calcium Binding ◽  
Metal Ion ◽  
Primary Event ◽  
Binding Properties ◽  
Physiological Concentration ◽  
Physiological Conditions ◽  
Sequential Mechanism

The results presented provide more information on the sequential mechanism that promotes the Ca2+-induced activation of human erythrocyte μ-calpain under physiological conditions. The primary event in this process corresponds to the binding of Ca2+ to eight interacting sites, of which there are four in each of the two calpain subunits. Progressive binding of this metal ion is linearly correlated with the dissociation of the proteinase, which reaches completion when all eight binding sites are occupied. The affinity for Ca2+ in the native heterodimeric calpain is increased 2-fold in the isolated 80 kDa catalytic subunit, but it reaches a Kd consistent with the physiological concentration of Ca2+ only in the active autoproteolytically derived 75 kDa form. Binding of Ca2+ in physiological conditions, and thus the formation of the 75 kDa subunit, can occur only in the presence of positive modulators. These are represented by the natural activator protein, found to be a Ca2+-binding protein, and by highly digestible substrates. The former produces a very large increase in the affinity of calpain for Ca2+, and the latter a smaller but still consistent decrease in the Kd of the proteinase for the metal ion. As a result, both dissociation into the constituent subunits and the autoproteolytic conversion of the native 80 kDa subunit into the active 75 kDa form can occur within the physiological fluctuations in Ca2+ concentration. The delay in the expression of the proteolytic activity with respect to Ca2+ binding to native calpain, no longer detectable in the 75 kDa form, can be attributed to a Ca2+-induced functional conformational change, which is correlated with the accessibility of the active site of the enzyme.

scholarly journals Regulatory effects of ATP and luciferin on firefly luciferase activity

1995 ◽  
Vol 305 (3) ◽  
pp. 929-933 ◽  
Author(s):  
N Lembert ◽  
L Å Idahl
Keyword(s):  
Steady State ◽  
Active Site ◽  
Luciferase Activity ◽  
Firefly Luciferase ◽  
Kinetic Properties ◽  
Allosteric Site ◽  
Experimental Conditions ◽  
Regulatory Effects ◽  
High Concentrations ◽  
Firefly Luciferase Activity

ATP and luciferin are not only substrates of firefly luciferase, but can, in addition, modulate its activity. High concentrations of luciferin induce a conformational change of the enzyme that temporarily reduces the catalytic rate. Re-activation takes approx. 20 min and is independent of variation in the concentration of enzyme or ATP, but lengthens with increasing luciferin concentration. High concentrations of albumin reduce this luciferin effect. The kinetic properties of firefly luciferase determined from initial rates and at steady state after 1 min of catalysis have been analysed according to Michaelis-Menten kinetics. There is only one active site for each of the substrates. At steady state the Km and Vmax. values for both substrates are reduced in an uncompetitive manner. Hyperbolic Lineweaver-Burk plots indicate an activation by ATP probably by binding to an allosteric site. A model is presented which incorporates luciferin induced de- and re-activation effects. Experimental conditions to avoid the regulatory effects of substrates during ATP monitoring are proposed.

scholarly journals Structural and catalytic effects of an invariant purine substitution in the hammerhead ribozyme: implications for the mechanism of acid–base catalysis

2014 ◽  
Vol 70 (9) ◽  
pp. 2256-2263 ◽  
Author(s):  
Eric P. Schultz ◽  
Ernesto E. Vasquez ◽  
William G. Scott
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Hammerhead Ribozyme ◽  
Base Catalysis ◽  
Metal Ion Binding ◽  
Acid Base ◽  
Kinetic Measurements ◽  
Structural Role ◽  
General Base

The hammerhead ribozyme catalyzes RNA cleavageviaacid–base catalysis. Whether it does so by general acid–base catalysis, in which the RNA itself donates and abstracts protons in the transition state, as is typically assumed, or by specific acid–base catalysis, in which the RNA plays a structural role and proton transfer is mediated by active-site water molecules, is unknown. Previous biochemical and crystallographic experiments implicate an invariant purine in the active site, G12, as the general base. However, G12 may play a structural role consistent with specific base catalysis. To better understand the role of G12 in the mechanism of hammerhead catalysis, a 2.2 Å resolution crystal structure of a hammerhead ribozyme fromSchistosoma mansoniwith a purine substituted for G12 in the active site of the ribozyme was obtained. Comparison of this structure (PDB entry 3zd4), in which A12 is substituted for G, with three previously determined structures that now serve as important experimental controls, allows the identification of structural perturbations that are owing to the purine substitution itself. Kinetic measurements for G12 purine-substituted schistosomal hammerheads confirm a previously observed dependence of rate on the pKaof the substituted purine; in both cases inosine, which is similar to G in pKaand hydrogen-bonding properties, is unexpectedly inactive. Structural comparisons indicate that this may primarily be owing to the lack of the exocyclic 2-amino group in the G12A and G12I substitutions and its structural effect upon both the nucleotide base and phosphate of A9. The latter involves the perturbation of a previously identified and well characterized metal ion-binding site known to be catalytically important in both minimal and full-length hammerhead ribozyme sequences. The results permit it to be suggested that G12 plays an important role in stabilizing the active-site structure. This result, although not inconsistent with the potential role of G12 as a general base, indicates that an alternative hammerhead cleavage mechanism involving specific base catalysis may instead explain the observed rate dependence upon purine substitutions at G12. The crystallographic results, contrary to previous assumptions, therefore cannot be interpreted to favor the general base catalysis mecahnism over the specific base catalysis mechanism. Instead, both of these mutually exclusive mechanistic alternatives must be considered in light of the current structural and biochemical data.

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