Pyruvate kinase of Leishmania mexicana mexicana Cloning and analysis of the gene, overexpression in Escherichia coli and characterization of the enzyme

Ernest, Isabelle; Callens, Mia; Opperdoes, Frederik; Michels, Paul A.M.

doi:10.1016/0166-6851(94)90133-3

Cited by 40 publications

(33 citation statements)

References 36 publications

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“…The major differences are listed in Table 4. In contrast, considerable conservation of the residues constituting the pocket was observed within the Trypanosomatidae T. brucei and Leishmania mexicana (as indicated in Table 4, as well as other residues not listed here) of which the PYKs are regulated in a highly similar manner (14,39). Particularly the bottom of the trypanosomal enzyme's pocket shows a major difference compared to that of the M1 PYK: Arg at position 22 instead of Gly (45 in the cat enzyme numbering).…”

Section: Modeling Of the Three-dimensional Structure Of T Brucei Pyrmentioning

confidence: 72%

Pyruvate Kinase ofTrypanosoma brucei:Overexpression, Purification, and Functional Characterization of Wild-Type and Mutated Enzyme

Ernest

Callens

Uttaro

et al. 1998

Protein Expression and Purification

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Section: Modeling Of the Three-dimensional Structure Of T Brucei Pyrmentioning

confidence: 72%

Pyruvate Kinase ofTrypanosoma brucei:Overexpression, Purification, and Functional Characterization of Wild-Type and Mutated Enzyme

Ernest

Callens

Uttaro

et al. 1998

Protein Expression and Purification

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“…Position 117 is occupied by Glu in 121 sequences and by Lys, Ser, and Arg in 106, 2, and 1 sequence, respectively. The requirement for K ϩ by 21 pyruvate kinases that have Glu at position 117 has been examined, and the activity of all of them is K ϩ -dependent (8,(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38). The activities of eight pyruvate kinases that do not have Glu at that position have been characterized; in all cases the activity is K ϩ -independent.…”

Section: Resultsmentioning

confidence: 99%

“…In addition, it has been shown that K ϩ changes the kinetic mechanism of rabbit muscle pyruvate kinase and is involved in the acquisition of the active conformation of the enzyme (20 (21), a water molecule, and a phosphate oxygen of phosphoenolpyruvate analogs (22), or an oxygen from the ␥-phosphate of ATP (5). For a long time, it was thought that the absolute dependence of K ϩ was a common feature to all pyruvate kinases (8,(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38). However, as the number of characterized enzymes increased, it became apparent that the activity of several pyruvate kinases was K ϩ -independent, for example those from Escherichia coli (Type II) (39), Phycomyces blakesleeanus (40), Corynebacterium glutamicum (41), Zymomonas mobilis (42), Thermoproteus tenax (43), Synechococcus pcc 6301 (44), Archaeoglobus fulgidus, Pyrobaculum aerophilum, Aeropyrum pernix, and Thermotoga maritima (45).…”

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confidence: 99%

Dichotomic Phylogenetic Tree of the Pyruvate Kinase Family

Oria-Hernández

Riveros‐Rosas

Ramírez‐Silva

2006

Journal of Biological Chemistry

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“…In mammals and many other species fructose 1,6-bisphosphate (Fru-1,6-BP) acts as the effector, but trypanosomatid PYKs are allosterically activated by fructose 2,6-bisphosphate (15). PYK is particularly suitable for studying allosteric regulation because unlike many other allosteric enzymes, the product does not bind to the effector site and participate in allosteric regulation.…”

mentioning

confidence: 99%

Allosteric Mechanism of Pyruvate Kinase from Leishmania mexicana Uses a Rock and Lock Model

Morgan

McNae

Nowicki

et al. 2010

Journal of Biological Chemistry

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131

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Allosteric regulation provides a rate management system for enzymes involved in many cellular processes. Ligand-controlled regulation is easily recognizable, but the underlying molecular mechanisms have remained elusive. We have obtained the first complete series of allosteric structures, in all possible ligated states, for the tetrameric enzyme, pyruvate kinase, from Leishmania mexicana. The transition between inactive T-state and active R-state is accompanied by a simple symmetrical 6 o rigid body rocking motion of the A-and C-domain cores in each of the four subunits. However, formation of the R-state in this way is only part of the mechanism; eight essential salt bridge locks that form across the C-C interface provide tetramer rigidity with a coupled 7-fold increase in rate. The results presented here illustrate how conformational changes coupled with effector binding correlate with loss of flexibility and increase in thermal stability providing a general mechanism for allosteric control.Allosteric regulation ("the second secret of life" quotation ascribed to Jacob Monod) (see Ref. 1) controls many important cellular processes, including signal transduction, transcription, and metabolism (2). It describes the effect of binding one ligand on the subsequent binding of a second ligand at a topographically distinct site. Human pyruvate kinase (hPYK) 2 provides a striking example of the significance of allosteric regulation: a splicing switch of the primary RNA transcript to yield the M1 or M2 isoenzymes is now known to be responsible for the Warburg effect in cancer (3, 4) and opens up the possibility of developing isoform specific therapeutics (5). Additional mechanisms of activity regulation, including the binding of amino acids, phosphorylation, and the binding of oncoproteins, may provide further therapeutic approaches for targeting hPYK (6, 7).Most allosterically regulated proteins are enzymes in which the binding of an activator or inhibitor to the effector site can affect the binding of a substrate at the active site. The MonodWyman-Changeux model of allostery (8) suggests that oligomeric enzymes undergo symmetrical transitions (classically between the T-and R-states) 3 (36) that can be stabilized by ligand binding. However, there are now examples of allosteric control that do not show obvious conformational change (9), and a growing body of work exists to support the idea that flexible regions of molecules (which exist as an ensemble of conformers) may undergo allosteric regulation by changes in the conformer population (10). There are examples of allosteric enzymes in which the same protein has been captured in both a T-state (which has low affinity for substrate) and an R-state (which has higher affinity for substrate), and some structural insight has been obtained by the study of at least one of the allosteric states of Ͼ50 proteins (10 -12). However, a full understanding of the allosteric effect requires structural information on each of four states: (i) apoenzyme, (ii) active site complex, (iii) eff...

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Pyruvate kinase of Leishmania mexicana mexicana Cloning and analysis of the gene, overexpression in Escherichia coli and characterization of the enzyme

Cited by 40 publications

References 36 publications

Pyruvate Kinase ofTrypanosoma brucei:Overexpression, Purification, and Functional Characterization of Wild-Type and Mutated Enzyme

Pyruvate Kinase ofTrypanosoma brucei:Overexpression, Purification, and Functional Characterization of Wild-Type and Mutated Enzyme

Dichotomic Phylogenetic Tree of the Pyruvate Kinase Family

Allosteric Mechanism of Pyruvate Kinase from Leishmania mexicana Uses a Rock and Lock Model

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