Rat intestinal trehalase. Studies of the active site.
Rat intestinal trehalase was solubilized, purified and reconstituted into proteoliposomes. With octyl glucoside as the solubilizing detergent, the purified protein appeared as a single band on SDS/polyacrylamide-gel electrophoresis with an apparent molecular mass of 67 kDa. Kinetic studies indicated that the active site of this enzyme can be functionally divided into two adjacent regions, namely a binding site (with pKa 4.8) and a catalytic site (with pKa 7.2). Other findings suggested that the catalytic site contains a functional thiol group, which is sensitive to inhibition by N-ethylmaleimide, Hg2+ and iodoacetate. Substrate protection and iodoacetate labelling of the thiol group demonstrated that only a protein of 67 kDa was labelled. Furthermore, sucrose and phlorizin protected the thiol group, but Tris-like inhibitors did not. Structure-inhibition analysis of Tris-like inhibitors, the pH effect of Tris inhibition and Tris protection of 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide inactivation permitted characterization and location of a separate site containing a carboxy group for Tris binding, which may also be the binding region. On the basis of these findings, a possible structure for the active site of trehalase is proposed.
Department of Medicine, Harvard Medical School, Boston, MA.
C C
CC
Chen
W J
WJ
Guo
K J
KJ
Isselbacher
D000818:Animals; D001665:Binding Sites; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007421:Intestine, Small / Q000201:enzymology*; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D008297:Male; D008956:Models, Chemical; D051381:Rats; D011919:Rats, Inbred Strains; D013329:Structure-Activity Relationship; D014198:Trehalase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D014325:Tromethamine / Q000494:pharmacology
D007461:Iodoacetates; D014325:Tromethamine; D014198:Trehalase; D019807:Iodoacetic Acid