Abstract
The transition between the inactive T-state (apoenzyme) and active R-state (effector bound enzyme) of Trypanosoma cruzi pyruvate kinase (PYK) is accompanied by a symmetrical 8° rigid body rocking motion of the A- and C-domain cores in each of the four subunits, coupled with the formation of additional salt bridges across two of the four subunit interfaces. These salt bridges provide increased tetramer stability correlated with an enhanced specificity constant (kcat/S0.5). A detailed kinetic and structural comparison between the potential drug target PYKs from the pathogenic protists T. cruzi, T. brucei and Leishmania mexicana shows that their allosteric mechanism is conserved. By contrast, a structural comparison of trypanosomatid PYKs with the evolutionarily divergent PYKs of humans and of bacteria shows that they have adopted different allosteric strategies. The underlying principle in each case is to maximize (kcat/S0.5) by stabilizing and rigidifying the tetramer in an active R-state conformation. However, bacterial and mammalian PYKs have evolved alternative ways of locking the tetramers together. In contrast to the divergent allosteric mechanisms, the PYK active sites are highly conserved across species. Selective disruption of the varied allosteric mechanisms may therefore provide a useful approach for the design of species-specific inhibitors.
Highlights
Pyruvate kinase (PYK, EC 2.7.1.40) catalyses the final step in glycolysis, namely the transfer of a phospho group from phosphoenolpyruvate (PEP) to ADP to form pyruvate and ATP.2014 The Authors
ADP, PEP, oxalate (OX), F16BP, F26BP, lactate dehydrogenase (LDH; rabbit muscle), polyethylene glycol (PEG) 8000, antibiotics and buffers were obtained from Sigma-Aldrich
Values of T. cruzi PYK (TcPYK) kinetic parameters were determined for PEP in the presence and absence of both the trypanosomatid pyruvate kinase (PYK) allosteric activator F26BP and the more general activator F16BP at 298 K and are summarized in table 1, together with the parameters previously reported for T. brucei PYK (TbPYK) [12]
Summary
PYK exists primarily as a homotetramer with subunits of 50–60 kDa (depending on species), each of which is composed of four domains: the N-terminal (not present in bacteria), A-, B- and C-domains (figure 1a). An additional C’-domain is found in PYKs from Geobacillus stearothermophilis [2] (figure 2) and Staphylococcus aureus [6]. The active site is nestled between the A- and B-domains and is located approximately 39 Å from the effector site which is located in the C-domain. The B-domain contributes a mobile lid at one end of the (α/β)8-barrelled A-domain and modulates access to the active site
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