RESUMO

Methanogenic archaea have received attention due to their potential use in biotechnological applications such as methane production, so their metabolism and regulation are topics of special interest. When growing in a nutrient-rich medium, these organisms exhibit gluconeogenic metabolism; however, under starvation conditions, they turn to glycolytic metabolism. To date, no regulatory mechanism has been described for this gluconeogenic/glycolytic metabolic switch. Here, we report that adenosine monophosphate (AMP) activates both enzymatic activities of the bifunctional adenosine diphosphate (ADP)-dependent phosphofructokinase/glucokinase from Methanococcus maripaludis (MmPFK/GK). To understand this phenomenon, we performed a comprehensive kinetic characterisation, including determination of the kinetics, substrate inhibition and AMP activation mechanism of this enzyme. We determined that MmPFK/GK has an ordered-sequential mechanism, in which MgADP is the first substrate to bind and AMP is the last product released. The enzyme also displays substrate inhibition by both sugar substrates; we determined that this inhibition occurs through the formation of catalytically nonproductive enzyme complexes caused by sugar binding. For both activities, the AMP activation mechanism occurs primarily through incremental changes in the affinity for the sugar substrate, with this effect being higher in the GK than in the PFK activity. Interestingly, due to the increase in the sugar substrate affinity caused by AMP, an enhancement in the sugar substrate inhibition effect was also observed for both activities, which can be explained by an increase in sugar binding leading to the formation of dead-end complexes. These results shed light on the regulatory mechanisms of methanogenic archaeal sugar metabolism, a phenomenon that has been largely unexplored.

Assuntos

Mathanococcus , Fosfofrutoquinases , Difosfato de Adenosina , Monofosfato de Adenosina , Mathanococcus/genética , Açúcares

RESUMO

During evolution, some homologs proteins appear with different connectivity between secondary structures (different topology) but conserving the tridimensional arrangement of them (same architecture). These events can produce two types of arrangements; circular permutation or non-cyclic permutations. The first one results in the N and C terminus transferring to a different position on a protein sequence while the second refers to a more complex arrangement of the structural elements. In ribokinase superfamily, two different topologies can be identified, which are related to each other as a non-cyclic permutation occurred during the evolution. Interestingly, this change in topology is correlated with the nucleotide specificity of its members. Thereby, the connectivity of the secondary elements allows us to distinguish an ATP-dependent and an ADP-dependent topology. Here we address the impact of introducing the topology of a homologous ATP-dependent kinase in an ADP-dependent kinase (Thermococcus litoralis glucokinase) in the structure, nucleotide specificity, and substrate binding order of the engineered enzyme. Structural evidence demonstrates that rewiring the topology of TlGK leads to an active and soluble enzyme without modifications on its three-dimensional architecture. The permuted enzyme (PerGK) retains the nucleotide preference of the parent TlGK enzyme but shows a change in the substrate binding order. Our results illustrate how the rearrangement of the protein folding topology during the evolution of the ribokinase superfamily enzymes may have dictated the substrate-binding order in homologous enzymes of this superfamily.

Assuntos

Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Glucoquinase/química , Glucoquinase/metabolismo , Estrutura Secundária de Proteína , Thermococcus/enzimologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Dicroísmo Circular , Cristalografia por Raios X , Cinética , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Dobramento de Proteína , Espalhamento a Baixo Ângulo , Especificidade por Substrato , Difração de Raios X

RESUMO

ABSTRACT The effectiveness of single site phase transfer catalyst- 2- benzoylethyldecyldimethylammonium bromide has been studied with the help of kinetics of free radical polymerization of butyl methacrylate. The radical polymerization was catalysed with single site phase transfer catalyst, initiated by water soluble potassium peroxydisulphate initiator in ethyl acetate / water biphasic media, under inert and unstirred condition at constant temperature, 60±1ºC. The dependence of rate of polymerization on various experimental conditions, different concentrations of monomer, initiator, and phase transfer catalyst was evaluated and effect of temperature as well as solvent polarity was determined. The order of the reaction with respect to monomer, initiator and phase transfer catalyst was found to be 1, 0.5 and 0.5 respectively. It was also observed that a slight increase in rate of polymerization as the polarity of solvents increased. A suitable kinetic mechanism has been suggested as per the investigational conditions. Molecular weight of the polybutyl methacrylate which has been polymerized bysingle site phase transfer catalyst was evaluated using gel permeation chromatographic technique.