New Insights on the Mechanism of the K⁺-Independent Activity of Crenarchaeota Pyruvate Kinases

<div><p>Eukarya pyruvate kinases have glutamate at position 117 (numbered according to the rabbit muscle enzyme), whereas in Bacteria have either glutamate or lysine and in Archaea have other residues. Glutamate at this position makes pyruvate kinases K⁺-dependent, whereas lysine confers K⁺-independence because the positively charged residue substitutes for the monovalent cation charge. Interestingly, pyruvate kinases from two characterized Crenarchaeota exhibit K⁺-independent activity, despite having serine at the equivalent position. To better understand pyruvate kinase catalytic activity in the absence of K⁺ or an internal positive charge, the <i>Thermofilum pendens</i> pyruvate kinase (valine at the equivalent position) was characterized. The enzyme activity was K⁺-independent. The kinetic mechanism was random order with a rapid equilibrium, which is equal to the mechanism of the rabbit muscle enzyme in the presence of K⁺ or the mutant E117K in the absence of K⁺. Thus, the substrate binding order of the <i>T</i>. <i>pendens</i> enzyme was independent despite lacking an internal positive charge. Thermal stability studies of this enzyme showed two calorimetric transitions, one attributable to the A and C domains (<i>T_m</i> of 99.2°C), and the other (<i>T_m</i> of 105.2°C) associated with the B domain. In contrast, the rabbit muscle enzyme exhibits a single calorimetric transition (<i>T_m</i> of 65.2°C). The calorimetric and kinetic data indicate that the B domain of this hyperthermophilic enzyme is more stable than the rest of the protein with a conformation that induces the catalytic readiness of the enzyme. B domain interactions of pyruvate kinases that have been determined in <i>Pyrobaculum aerophilum</i> and modeled in <i>T</i>. <i>pendens</i> were compared with those of the rabbit muscle enzyme. The results show that intra- and interdomain interactions of the Crenarchaeota enzymes may account for their higher B domain stability. Thus the structural arrangement of the <i>T</i>. <i>pendens</i> pyruvate kinase could allow charge-independent catalysis.</p></div

Conservation of the K⁺ binding site residues and of those that covariate with the corresponding position 117 in Crenarchaeota subdomain.

<p>In <b>A</b>. the subtree of the Crenarchaeota branch that contains the <i>Tp</i>PK sequence is shown. The unrooted phylogenetic tree that includes all available PK protein sequences from the Archaea domain is shown in Supplemental data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119233#pone.0119233.s001" target="_blank">S1 Fig</a>.): here, only the subtree of the Crenarchaeota branch that included <i>Tp</i>PK is presented. Residues 113, 114, 117, and 120 (according to RMPK numbering), as well as the accession numbers are indicated. The branches are not drawn to scale, and only the branch topology is shown. <b>B</b>. Logos of the K⁺ binding site residues in PK from animals and from the Crenarchaeota subdomain are presented. In animals, the numbering is according to RMPK, and the equivalent positions in the Crenarchaeota subdomain are given according to <i>Tp</i>PK numbering. <b>C</b>. The K⁺ binding site in RMPK (PDBID 1A49, subunit B) and hypothetical residues of coordination to the monovalent cation in <i>Pa</i>PK (PDBID 3QTG). In RMPK, K⁺ is shown in purple and the distances from K⁺ to Oγ of Ser76, O∂1 of Asn74, O∂1 of Asp112 and the carbonyl oxygen of Thr113 are 3.1, 2.6, 2.6 and 2.8 Å, respectively (not shown). The distances shown are those between the coordination residues and from O∂2 of Glu117 to K⁺ (RMPK) or from Oγ of Ser85 to Cß of Ala50 and the carbonyl oxygen of Leu81 (<i>Pa</i>PK).</p

Dead-end inhibition patterns and inhibition constants for oxalate and AMP in <i>Tp</i>PK.

<p>The experimental conditions were as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119233#pone.0119233.g002" target="_blank">Fig. 2</a>. The reciprocals of the concentrations of ionized PEP and ADP-Mg complexes are shown in the abscissas of each graph. In plot <b>A</b>, the concentrations of PEP^3- were 0.054, 0.077, 0.10, 0.22, and 1.1 mM. The Mg²⁺_free and ADP concentrations were kept constant at 30 mM and 2 mM, respectively. The fixed concentrations of oxalate were 0 (∎), 10 (●), 20 (▴), 30 (▾) and 40 (◆) μM. In plot <b>B</b>, the concentrations of the ADP-Mg complexes were 0.045, 0.063, 0.090, 0.18, and 0.90 mM. The ionized PEP concentration was kept constant at 30 mM. The Mg²⁺_free and oxalate concentrations were as in plot A. In plot <b>C</b>, the concentrations of PEP^3- were 0.15, 0.30, 0.55, 0.77, and 1.5 mM. The Mg²⁺_free and ADP concentrations were as in plot A. The fixed concentrations of AMP were 0 (∎), 4 (●), 8 (▴), 12 (▾) and (◆) 16 mM. In plot <b>D</b>, the concentrations of ADP were as in plot <i>B</i>. The Mg²⁺_free and ionized PEP concentrations were kept constant at 30 mM. The concentrations of AMP were 0 (∎), 8 (●), 12 (▴), 16(▾) and 20 (◆) mM.</p

Models of the hydrophobic core of the B domain of RMPK (A), <i>Pa</i>PK (B) and <i>Tp</i>PK (C). Interactions between domains A and B of RMPK (D) and <i>Pa</i>PK (E).

<p>The aromatic residues at the hydrophobic core are represented as sticks. The residues involved in polar and hydrophobic interactions between domain A and domain B are shown as sticks. The red dotted lines highlight polar interactions. Notice that these interactions are absent in RMPK. This figure was constructed from the coordinates of RMPK and <i>Pa</i>PK deposited under file names 2G50 and 3QTG at the PDB. It is noteworthy that the interdomain interactions were not analyzed in the modeled monomer of <i>Tp</i>PK but were determined from the structure of <i>Pa</i>PK to obtain reliable results.</p

Intersecting patterns, kinetic mechanisms, and kinetic constants for <i>Tp</i>PK.

<p>Intersecting patterns were taken from the double reciprocal plots of the initial velocity data. The data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119233#pone.0119233.g002" target="_blank">Fig. 2</a> were globally fit to the equation describing a rapid-equilibrium random-order mechanism as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119233#pone.0119233.t001" target="_blank">Table 1</a>. The specificity coefficients <i>k</i>_cat/<i>K</i>_<i>m</i> (M^-1 s^-1) are expressed in log form.</p><p>Intersecting patterns, kinetic mechanisms, and kinetic constants for <i>Tp</i>PK.</p

Loss of native contacts (Q_Cα) <i>versus</i> time for the modeled <i>Tp</i>PKs and RMPKs at 500 K.

<p>The native contacts for the PK monomer and of the A, B and C domains are shown. <i>Tp</i>PKs and RMPKs correspond to panels <b>A</b> through <b>D</b> and to panels <b>E</b> through <b>H</b>, respectively. Simulations were performed for 50 ns in an implicit solvent at 500 K. The simulations were started from two different conformations of the enzymes, open (black lines) and closed (grey lines). The open and closed conformations for <i>Tp</i>PK were modeled as described in Material and Methods. The open and closed conformations for RMPK were obtained for PDBID 2G50 and PDBID 1A5U, respectively. The simulations were run in triplicate. Although the simulations were run for 10 to 50 ns, only the relevant periods for the transitions are shown.</p

Double reciprocal plots from the initial velocity data of the <i>Tp</i>PK reaction.

<p>The reaction medium consisted of 3 ml of 50 mM Tris-HCl pH 6.0 containing 0.24 mM NADH, 30 mM Mg²⁺_free, and 8 μg/ml LDH. The reciprocals of the concentrations of ionized PEP and ADP-Mg complexes are shown in the <i>abscissas</i> of each graph. <b>A</b>. The fixed concentrations of ADP-Mg were 0.084 (■), 0.12 (●), 0.16 (▲), 0.37 (▼), and 0.67 mM (♦). <b>B</b>. The fixed concentrations of PEP^3- were 0.031 (■), 0.14 (●), 0.34 (▲), 0.69(▼), and 1.30 mM (♦). The Mg²⁺_free concentration was kept constant at 30 mM. To maintain the ionic strength, (CH₃)₄NCl was added to a final salt concentration of 0.25 M. The reaction was started with the addition of PK. The concentrations of PK were 0.32 and 0.16 μg/ml for the three lowest and two highest substrate concentrations, respectively.</p

Differential scanning calorimetry of the <i>Tp</i>PK.

<p><b>A</b>. <i>Tp</i>PK without and with 0.2 mM Mn²⁺ are represented by solid and dashed-dotted lines, respectively. <b>B</b>. <i>Tp</i>PK: solid line, RMPK: dashed line, <b>C</b>. <i>Tp</i>PK: solid line, the B domain: dotted line. The enzyme concentration was 1.0 mg/ml (19.47 μM monomer) for <i>Tp</i>PK, the B domain concentration was 0.18 mg/ml (19.55 μM of domain). The scan rate was 1.5°C/min.</p

Structural Effects of Protein Aging: Terminal Marking by Deamidation in Human Triosephosphate Isomerase

<div><p>Deamidation, the loss of the ammonium group of asparagine and glutamine to form aspartic and glutamic acid, is one of the most commonly occurring post-translational modifications in proteins. Since deamidation rates are encoded in the protein structure, it has been proposed that they can serve as molecular clocks for the timing of biological processes such as protein turnover, development and aging. Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins. Here, we studied the effects of deamidation on human triosephosphate isomerase (HsTIM), an enzyme for which deamidation of N15 and N71 has been long recognized as the signal for terminal marking of the protein. Deamidation was mimicked by site directed mutagenesis; thus, three mutants of HsTIM (N15D, N71D and N15D/N71D) were characterized. The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme. In contrast, the N15D mutant displays all the detrimental effects related to deamidation. The N15D/N71D mutant shows only minor additional effects when compared with the N15D mutation, supporting that deamidation of N71 induces negligible effects. The crystal structures show that, in contrast to the N71D mutant, where minimal alterations are observed, the N15D mutation forms new interactions that perturb the structure of loop 1 and loop 3, both critical components of the catalytic site and the interface of HsTIM. Based on a phylogenetic analysis of TIM sequences, we propose the conservation of this mechanism for mammalian TIMs.</p></div

Structural and Functional Perturbation of <i>Giardia lamblia</i> Triosephosphate Isomerase by Modification of a Non-Catalytic, Non-Conserved Region

<div><p>Background</p><p>We have previously proposed triosephosphate isomerase of <i>Giardia lamblia</i> (GlTIM) as a target for rational drug design against giardiasis, one of the most common parasitic infections in humans. Since the enzyme exists in the parasite and the host, selective inhibition is a major challenge because essential regions that could be considered molecular targets are highly conserved. Previous biochemical evidence showed that chemical modification of the non-conserved non-catalytic cysteine 222 (C222) inactivates specifically GlTIM. The inactivation correlates with the physicochemical properties of the modifying agent: addition of a non-polar, small chemical group at C222 reduces the enzyme activity by one half, whereas negatively charged, large chemical groups cause full inactivation.</p><p>Results</p><p>In this work we used mutagenesis to extend our understanding of the functional and structural effects triggered by modification of C222. To this end, six GlTIM C222 mutants with side chains having diverse physicochemical characteristics were characterized. We found that the polarity, charge and volume of the side chain in the mutant amino acid differentially alter the activity, the affinity, the stability and the structure of the enzyme. The data show that mutagenesis of C222 mimics the effects of chemical modification. The crystallographic structure of C222D GlTIM shows the disruptive effects of introducing a negative charge at position 222: the mutation perturbs loop 7, a region of the enzyme whose interactions with the catalytic loop 6 are essential for TIM stability, ligand binding and catalysis. The amino acid sequence of TIM in phylogenetic diverse groups indicates that C222 and its surrounding residues are poorly conserved, supporting the proposal that this region is a good target for specific drug design.</p><p>Conclusions</p><p>The results demonstrate that it is possible to inhibit species-specifically a ubiquitous, structurally highly conserved enzyme by modification of a non-conserved, non-catalytic residue through long-range perturbation of essential regions.</p></div