Nowadays there is no information if bifunctionality is an

Nowadays, there is no information if bifunctionality is an exclusive trait of the order Methanococcales or if this characteristic is shared by other orders from archaea, like Methanosarcinales. These organisms are mainly methanogenic, and are not able to use exogenous hexoses as carbon source for cell growth, although some of them have the ability to accumulate glycogen. For example, in the case of Methanosarcina acetivorans, glycogen storage was used as an MMP-2 Inhibitor I source through glycolysis to yield pyruvate, which in turn can be metabolized to give, among others, a methyl group for methane production and ATP synthesis [14]. In this context the presence of active glycolytic enzymes is essential. Since the genomes from many organisms from the order Methanosarcinales are available we decided to analyze them and assess if the bifunctional trait is a common characteristic of the enzymes from this order. Kinetic characterization of the ADP-dependent kinase from M. burtonii and from M. evestigatum demonstrated that both enzymes are bifunctional. Moreover, through an evolutive analysis we determine that there is a high conservation of active site residues in all the PFK enzymes from the order Methanosarcinales. Based on this information, all the enzymes should be bifunctional, as was described previously for the order Methanococcales [13], supporting the redundancy of the GK activity in the order Methanosarcinales. Analyses of structural information, as well as homology models for ADP-dependent kinases from different orders, especially regarding residues responsible for sugar substrate interactions, allowed us to establish a sequence signature for sugar substrate specificity that identifies specific GK or PFK, and bifunctional enzymes in this family. Understanding the kinetic characteristics of enzymes involved in glycolysis/gluconeogenesis in the order Methanosarcinales, may help to optimize and increase methane production, which has ecological and biotechnological relevance.
Materials and methods
Results and discussion
Acknowledgements This work was supported by Fondo Nacional de Desarrollo Científico y Tecnológico (Fondecyt 1150460).
Introduction The classic view of allosteric regulation, centering on ligand - mediated shifts in interaction between multiple protein subunits (e.g. positive cooperativity in hemoglobin), was formulated around proteins that were multimeric with the consideration of conformational transitions due to equilibrium ligand binding [1]. However, based on a growing number of monomeric enzyme systems that display various forms of cooperativity, one must consider a broader definition of allostery, which includes how binding events at one location in a protein (or ligand-protein complex) affect dynamics, catalysis and the distribution of conformations at remote locations [2]. In contrast to the classic allosteric theories, which considered that the ligand induced conformational transitions are faster than the catalytic steps, the “mnemonical” model and the “slow transition” model have been proposed to explain the apparent positive cooperativity of monomeric proteins [3]. Remarkably, monomeric allostery arises from conformational transitions that occur at a pace that is no longer fast in comparison to the rate of the catalytic reaction, kcat. It is exceedingly challenging to elucidate the source of catalytic enhancement (or inhibition) due to remote binding of allosteric effectors, where there is a lack of atomistic structural information; one of the paradigms for revealing the novel and broader principles of allostery is characterized by the monomeric human glucokinase (GK). Glucokinase (GK, also known as hexokinase IV) catalyzes the phosphorylation of glucose to glucose-6-phosphate (G6P) in the liver and the pancreas [4]. In hepatocytes, GK catalyzes the rate-determining step in glycolysis; while in pancreatic β-cells, it is involved in controlling insulin release [5]. GK has a relatively high KM for glucose and exhibits an apparent positive cooperativity as indicated by a hill coefficient of 1.7, which identifies it as a unique member of the hexokinase family [6]. It is believed that the distinct kinetic characteristics of GK forms account for its role as a sensor of glucose metabolism and homeostasis. Human GK has attracted much research interest as a novel drug target for type 2 diabetes (T2D) in recent years [7, 8]. Small-molecule allosteric activators of GK (GKA) were effective in lowering plasma glucose levels, increasing insulin secretion and decreasing hepatic glucose yield in T2D patients [9, 10], but recent clinical studies suggested that GKAs administration corresponded with an increase of hypoglycemia occurrence [10].