Differential mechanism-based labeling and unequivocal activity assignment of the two active sites of intestinal lactase/phlorizin hydrolase

Milk lactose is hydrolysed to galactose and glucose in the small intestine of mammals by the lactase/phlorizin hydrolase complex (LPH; EC 3.2.1.108/62 ). The two enzymatic activities, lactase and phlorizin hydrolase, are locat ed in the same polypeptide chain. According to sequence homology, mature LP H contains two different regions (III and IV), each of them homologous to f amily 1 glycosidases and each with a putative active site. There has been s ome discrepancy with regard to the assignment of enzymatic activity to the two active sites. Here we show differential reactivity of the two active si tes with mechanism-based glycosidase inhibitors. When LPH is treated with 2 ',4'-dinitrophenyl 2-deoxy-2-fluoro-beta -D-glucopyranoside (1) and 2',4'-d initrophenyl-2-deoxy-2-fluoro-beta -D-galactopyranoside (2), known mechanis m-based inhibitors of glycosidases, it is observed that compound 1 preferen tially inactivates the phlorizin hydrolase activity whereas compound 2 is s elective for the lactase active site. On the other hand, glycals (D-glucal and D-galactal) competitively inhibit lactase activity but not phlorizin hy drolase activity. This allows labeling of the phlorizin site with compound 1 by protection with a glycal. By differential labeling of each active site using 1 and 2 followed by proteolysis and MS analysis of the labeled fragm ents, we confirm that the phlorizin hydrolysis occurs mainly at the active site located at region III of LPH and that the active site located at regio n IV is responsible for the lactase activity. This assignment is coincident with that proposed from the results of recent active-site mutagenesis stud ies [Zecca, L., Mesonero, J.E., Stutz, A., Poiree, J.C., Giudicelli, J., Cu rsio, R., Gloor, S.M. & Semenza, G. (1998) FEES Lett. 435, 225-228] and opp osite to that based on data from early affinity labeling with conduritol B epoxide [Wacker, W., Keller, P., Falchetto, R., Legler, G. & Semenza, G. (1 992).