Verification of the lactase site of rat lactase-phlorizin hydrolase by site-directed mutagenesis.

Abstract:

BACKGROUND & AIMS:Lactase-phlorizin hydrolase (LPH) is an intestinal microvillus membrane glycoprotein that hydrolyzes lactose and phlorizin. These enzymatic activities have been assigned to glutamic acid (E) residues 1271 and 1747 in rabbit LPH. The aim of this study was to determine directly if this assignment was correct and if these two amino acids are the only nucleophiles required for LPH enzyme activity. METHODS:Site-directed mutagenesis of a full-length rat LPH complementary DNA was used to convert the rat homologues E1274 and E1750 to aspartic acid or glycine. Mutants were analyzed by enzyme activity assays. RESULTS:All tested activities of E1274D and E1274G were virtually unaffected. In contrast, mutations E1750D and E1750G resulted in total loss of lactase and cellobiose activities, leaving only low ONP-glc and ONP-gal hydrolase activities detectable. A double mutant containing both E1274G and E1750G had no activity. CONCLUSIONS:These studies directly confirm that the two previously identified glutamic acids are essential to the enzymatic activity of rat LPH. Rat lactase activity is not associated with the E1274 site. This study provides the first evidence that rat LPH has its major catalytic site at E1750, representing all of the lactase and the majority of the phlorizin hydrolase activity.

journal_name

Gastroenterology

journal_title

Gastroenterology

authors

Neele AM,Einerhand AW,Dekker J,Büller HA,Freund JN,Verhave M,Grand RJ,Montgomery RK

doi

10.1016/0016-5085(95)90583-9

subject

Has Abstract

pub_date

1995-10-01 00:00:00

pages

1234-40

issue

4

eissn

0016-5085

issn

1528-0012

pii

0016-5085(95)90583-9

journal_volume

109

pub_type

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