Molecular crowding modulates enzymatic activity and structure of -Gal from Kluyveromices lactis.

NOE, MELANIA MACARENA; CLOP, PEDRO DIEGO; PERILLO, MARÍA A.; NOLAN, VERONICA

La Plata

Congreso; Reunión Anual de la Sociedad ARgentina de Biofisica; 2018

Sociedad Argentina de Biofisica

-D-galactosidase [EC 3.2.1.23] (-Gal) is a soluble enzyme capable of catalyzing lactose hydrolysis into its constitutive monosaccharides: glucose and galactose. -Gal has been extensively studied because of its nutritional, biotechnological and therapeutic impact. Not only in the cellular milieu but also in situations of technological interest like during encapsulation, the activity of -Gal has to be evaluated in crowded systems.-Gal from kluyveromices lactis was historically isolated as a dimeric active protein of about 250 kDa. But, recent researches have described that in some conditions, like those generated by the presence of crowding conditions in the interior of a non-denaturing electrophoresis gel, a tetrameric form appear. This tetramer has lower activity than the dimeric state.The objective of the present work was to evaluate the effect of molecular crowding on -Gal structure and the relationship with its enzymatic activity modulation. PEG6000, a non-charged highly water-soluble polymer with well-known effects on water dynamics was used to produce the crowded environment. The enzymatic reaction was evaluated by measuring kinetic parameters of -Gal against an artificial substrate (ONPG). Protein conformation and thermal stability were analysed by fluorescence spectroscopy. Results showed that molecular crowding induced changes on kinetic parameters of -Gal: at low molecular crowding agent concentration, an enhancement on enzymatic activity was observed, while at high crowding agent concentration, a qualitative change from a michaelian to a sigmoidal behavior was observed. The studies on protein conformation showed that molecular crowding affected -Gal structure: changes on fluorescence emission spectra and protein calorimetric profile were observed. Changes in protein compactness and hydration could be the responsible for the qualitative change behaviour observed at the highest molecular crowding assayed.Acknowledgment: to SeCyT, CONICET and Foncyt for financial support. MAP and MVN are members of CIC from CONICET. MMN has a fellowship from CONICET.