Increased Stability and Catalytic Efficiency of Yeast Hexokinase Upon Interaction with Zwitterionic Micelles. Kinetics and Conformational Studies

Abstract

The effect of ligands (glucose, ATP and Mg2+) and zwitterionic micelles of lysophosphatidylcholine (LPC) or N-hexadecyl-N,N-dimethyl-3-ammonium propanesulfonate (HPS) in the yeast hexokinase (HK) stability was studied at 35 degrees C. The thermal inactivation kinetics followed one-exponential decay. The effect of ligands on protecting the enzyme against inactivation followed the order: glucose > glucose/Mg2+ >ATP/Mg2+ approximately or approximately equal to Mg2+l approximately or approximately equal to buffer only. Both LPC and HPS micelles increased the enzyme stability only when the incubation medium contained glucose or glucose/Mg2+, suggesting that the protein conformation is a key prerequisite for the enzyme-micelle interaction to take place. This enzyme-micelle interaction resulted in an increased catalytic efficiency (with a decrease in Km for ATP and increase in Vmax as well as in changes on the tertiary (intrinsic fluorescence) structure of the yeast hexokinase.

Superactivity and conformational changes on alpha-chymotrypsin upon interfacial binding to cationic micelles

“…Therefore, the observed effects depend on the type of interaction, which can be modulated by changes in pH or ionic strength. These studies with different enzymes showed a correlation between the changes in the kinetic and some conformational property of these proteins as studied by fluorescence [17][18][19], UVdifference [17] or ESR [16] spectroscopy.…”

“…The activity and stability of soluble enzymes in aqueous micelles is closely related to the chemical nature of the surfactant [16][17][18][19][20]. Therefore, the observed effects depend on the type of interaction, which can be modulated by changes in pH or ionic strength.…”

Effects of head group of cationic surfactants on the hydrolysis of p‐nitrophenyl acetate catalyzed by α‐chymotrypsin

“…The increase in head group size or hydrophilicity of surfactant play a significant role on the superactivity of α‐CT and hydrophobic chain promotes additives–enzyme interactions. The influence of additives on the catalytic properties of enzymes strongly depends on the additive structure, that is, the charge and the size of the head group and on the nature of the substrate 19–23. Alfani and coworkers 24–27 reported a positive correlation between the superactivity of α‐CT and the surfactant hydrophobicity.…”

Titration Calorimetry as a Tool to Determine Thermodynamic and Kinetic Parameters of Enzymes

“…Hexokinases catalyse the transfer of a phosphoryl group from ATP to hexoses, where the reaction is usually studied by a coupled enzyme assay with glucose-6-phosphate dehydrogenase. 20 Since hexokinases use two substrates, glucose and ATP, in the reaction, it is essential to use limited amounts of one substrate, maintaining the other in excess in order to find Q T and, consequently, DH cal . This is necessary since one of the substrates should be totally consumed to obtain DH cal .…”

“…It is also possible to use a coupled enzyme assay in order to study one particular reaction in which the product can be used as a reactant by an additional enzyme. 20,21 The disadvantage of this method is the need for large amounts of the additional enzyme and the required cofactors to avoid unintended errors in the characterization of the enzyme of interest.…”