Identification of Essential Residues for Catalysis of Rat Intestinal Phospholipase B/Lipase

详细信息    查看全文

• 作者：Ting Lu ; Masafumi Ito ; Urbain Tchoua ; Hiroshi Takemori ; Mitsuhiro Okamoto ; and Hiromasa Tojo
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：June 19, 2001
• 年：2001
• 卷：40
• 期：24
• 页码：7133 - 7139
• 全文大小：86K
• 年卷期：v.40,no.24(June 19, 2001)
• ISSN：1520-4995

文摘

Intestinal brush border membrane-associated phospholipase B/lipase (PLB/LIP) consists offour tandem homologous domains (repeats 1 through 4) and a COOH-terminal membrane binding domain,and repeat 2 is the catalytic domain that catalyzes phospholipase A₂, lysophospholipase, and lipase activities.We examined the structural basis of the catalysis of PLB/LIP with this unique substrate specificity bysite-directed mutagenesis of recombinant repeat 2 enzyme. Ser⁴¹⁴ and Ser⁴⁵⁹ within the active serine-containing consensus sequence G-X-S-X-G in the best-established lipase family were dispensablefor activity. In contrast, substitution of Ala for Ser⁴⁰⁴ almost completely inactivated the three lipolyticactivities of PLB/LIP, even though the gross conformation was not altered as determined by CDspectroscopy. Notably, this Ser is located within the conserved G-D-S-L sequence on the NH₂-terminalside in lipolytic enzymes of another group proposed recently. Furthermore, mutagenesis and CDspectroscopic analyses suggested that Asp⁵¹⁸ and His⁶⁵⁹, lying within conserved short stretches in thelatter group of lipolytic enzymes, were essential for activity. These three essential residues are conservedin the known PLB/LIP enzymes, suggesting that they form the catalytic triad in the active site. Theseresults indicate that PLB/LIP represents a distinct class of the lipase family. PLB/LIP is the first mammalianmember of that family. Repeat 2 is equipped with the triad, but not the other repeats, accounting for whyonly repeat 2 is the catalytic domain. Replacing Thr⁴⁰⁶ with Gly, matching the enzyme's sequence to thelipase consensus sequence exactly, led to a great decrease in secretion and accumulation of inactive enzymein the cells, suggesting a role of Thr⁴⁰⁶ in the structural stability.