Identification of essential residues for catalysis of rat intestinal phospholipase B/lipase

Intestinal brush border membrane-associated phospholipase B/lipase (PLB/LIP ) consists of four tandem homologous domains (repeats 1 through 4) and a CO OH-terminal membrane binding domain, and repeat 2 is the catalytic domain t hat catalyzes phospholipase A(2), lysophospholipase, and lipase activities. We examined the structural basis of the catalysis of PLB/LIP with this uni que substrate specificity by site-directed mutagenesis of recombinant repea t 2 enzyme. Ser(414) and Ser(459) within the active serine-containing conse nsus sequence G-X-S-X-G in the best-established lipase family were dispensa ble for activity. In contrast, substitution of Ala for Ser(404) almost comp letely inactivated the three lipolytic activities of PLB/LIP, even though t he gross conformation was not altered as determined by CD spectroscopy. Not ably, this Ser is located within the conserved G-D-S-L sequence on the NH2- terminal side in lipolytic enzymes of another group proposed recently. Furt hermore, mutagenesis and CD spectroscopic analyses suggested that Asp(518) and His(659), lying within conserved short stretches in the latter group of lipolytic enzymes, were essential for activity. These three essential resi dues are conserved in the known PLB/LIP enzymes, suggesting that they form the catalytic triad in the active site. These results indicate that PLB/LIP represents a distinct class of the lipase family. PLB/LIP is the first mam malian member of that family. Repeat 2 is equipped with the triad, but mot the other repeats, accounting for why only repeat 2 is the catalytic domain . Replacing Thr(406) with Gly, matching the enzyme's sequence to the lipase consensus sequence exactly, led to a great decrease in secretion and accum ulation of inactive enzyme in the cells, suggesting a role of Thr(406) in t he structural stability.