Verification of the lactase site of rat lactase-phlorizin hydrolase by site-directed mutagenesis.
OBJECTIVE
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.
Neele
A M
AM
Division of Pediatric Gastroenterology and Nutrition, Floating Hospital for Children, New England Medical Center Hospitals, Boston, Massachusetts, USA.
Einerhand
A W
AW
Dekker
J
J
Büller
H A
HA
Freund
J N
JN
Verhave
M
M
Grand
R J
RJ
Montgomery
R K
RK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D043322:Lactase; D010694:Lactase-Phlorizin Hydrolase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011233:Precipitin Tests; D011817:Rabbits; D051381:Rats; D014162:Transfection; D001616:beta-Galactosidase / Q000032:analysis*
D043322:Lactase; D001616:beta-Galactosidase; D010694:Lactase-Phlorizin Hydrolase