The Importance of Polarity in the Evolution of the K+ Binding Site of Pyruvate Kinase

Ramírez‐Silva, Leticia; Guerrero-Mendiola, Carlos; Cabrera, Nallely

doi:10.3390/ijms151222214

Cited by 2 publications

(7 citation statements)

References 32 publications

(40 reference statements)

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“…The kinetic parameters for the activation of Vc IPK by K + , NH 4 + , Rb + and Cs + are shown in Table 1 , but kinetic parameters for Na + and Li + could not to be calculated, because the lack of saturation to include in Table 1 . In comparison with other K + -dependent PKs, Vc IPK exhibited higher k cat and 2 to 5-fold lower K 0.5 for the monovalent cations [ 34 , 40 ]. According to [ 41 , 42 ], VCIPK is a good example of Type Ib activation by monovalent cations.…”

Section: Resultsmentioning

confidence: 99%

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The contribution of two isozymes to the pyruvate kinase activity of Vibrio cholerae: One K+-dependent constitutively active and another K+-independent with essential allosteric activation

Guerrero-Mendiola¹,

García-Trejo²,

Encalada³

et al. 2017

PLoS ONE

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In a previous phylogenetic study of the family of pyruvate kinase EC (2.7.1.40), a cluster with Glu117 and another with Lys117 were found (numbered according to the rabbit muscle enzyme). The sequences with Glu117 have been found to be K+-dependent, whereas those with Lys117 were K+-independent. Interestingly, only γ-proteobacteria exhibit sequences in both branches of the tree. In this context, it was explored whether these phylogenetically distinct pyruvate kinases were both expressed and contribute to the pyruvate kinase activity in Vibrio cholerae. The main findings of this work showed that the isozyme with Glu117 is an active K+-dependent enzyme. At the same substrate concentration, its Vmax in the absence of fructose 1,6 bisphosphate was 80% of that with its effector. This result is in accordance with the non-essential activation described by allosteric ligands for most pyruvate kinases. In contrast, the pyruvate kinase with Lys117 was a K+-independent enzyme displaying an allosteric activation by ribose 5-phosphate. At the same substrate concentration, its activity without the effector was 0.5% of the one obtained in the presence of ribose 5-phosphate, indicating that this sugar monophosphate is a strong activator of this enzyme. This absolute allosteric dependence is a novel feature of pyruvate kinase activity. Interestingly, in the K+-independent enzyme, Mn2+ may “mimic” the allosteric effect of Rib 5-P. Despite their different allosteric behavior, both isozymes display a rapid equilibrium random order kinetic mechanism. The intracellular concentrations of fructose 1,6-bisphosphate and ribose 5-phosphate in Vibrio cholerae have been experimentally verified to be sufficient to induce maximal activation of both enzymes. In addition, Western blot analysis indicated that both enzymes were co-expressed. Therefore, it is concluded that VcIPK and VcIIPK contribute to the activity of pyruvate kinase in this γ-proteobacterium.

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Section: Resultsmentioning

confidence: 99%

“…In comparison with other K + -dependent PKs, Vc IPK exhibited higher k cat and 2 to 5-fold lower K 0.5 for the monovalent cations [34, 40]. According to [41,42], VCIPK is a good example of Type Ib activation by monovalent cations.…”

Section: Resultsmentioning

confidence: 99%

The contribution of two isozymes to the pyruvate kinase activity of Vibrio cholerae: One K+-dependent constitutively active and another K+-independent with essential allosteric activation

Guerrero-Mendiola¹,

García-Trejo²,

Encalada³

et al. 2017

PLoS ONE

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“…It is important to note that the activation curves of WT-RMPK with Na + and Li + did saturate (see Table 1). In previous studies, WT-RMPK activation exhibited no saturation of these cations at an ionic strength of 223 mM ionic strength maintained with HEPES [21]. (CH 3 ) 4 N + may bind to non-specific sites of the enzyme favoring Na + and Li + occupation of the monovalent binding site.…”

Section: Resultsmentioning

confidence: 81%

“…In contrast, ion selectivity of T113L was altered; maximal activation followed the order Rb + > NH 4 + > K + > Cs + . It was found that the polarity of residue 113 is determinant in the partition of K + into its site [21]. E117K (Figure 4B), T113L/E117K (Figure 4C), and T113L/K114Q/E117K mutants (Figure 4D) were K + -independent.…”

Section: Resultsmentioning

confidence: 97%

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The K+-Dependent and -Independent Pyruvate Kinases Acquire the Active Conformation by Different Mechanisms

Ramírez‐Silva

Hernández-Alcántara

Guerrero-Mendiola

et al. 2022

IJMS

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Eukarya pyruvate kinases possess glutamate at position 117 (numbering of rabbit muscle enzyme), whereas bacteria have either glutamate or lysine. Those with E117 are K+-dependent, whereas those with K117 are K+-independent. In a phylogenetic tree, 80% of the sequences with E117 are occupied by T113/K114/T120 and 77% of those with K117 possess L113/Q114/(L,I,V)120. This work aims to understand these residues’ contribution to the K+-independent pyruvate kinases using the K+-dependent rabbit muscle enzyme. Residues 117 and 120 are crucial in the differences between the K+-dependent and -independent mutants. K+-independent activity increased with L113 and Q114 to K117, but L120 induced structural differences that inactivated the enzyme. T120 appears to be key in folding the protein and closure of the lid of the active site to acquire its active conformation in the K+-dependent enzymes. E117K mutant was K+-independent and the enzyme acquired the active conformation by a different mechanism. In the K+-independent apoenzyme of Mycobacterium tuberculosis, K72 (K117) flips out of the active site; in the holoenzyme, K72 faces toward the active site bridging the substrates through water molecules. The results provide evidence that two different mechanisms have evolved for the catalysis of this reaction.

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The Importance of Polarity in the Evolution of the K+ Binding Site of Pyruvate Kinase

Cited by 2 publications

References 32 publications

The contribution of two isozymes to the pyruvate kinase activity of Vibrio cholerae: One K+-dependent constitutively active and another K+-independent with essential allosteric activation

The contribution of two isozymes to the pyruvate kinase activity of Vibrio cholerae: One K+-dependent constitutively active and another K+-independent with essential allosteric activation

The K+-Dependent and -Independent Pyruvate Kinases Acquire the Active Conformation by Different Mechanisms

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