Identification of Asp 549 as the Catalytic Nucleophile of Glycogen-Debranching Enzyme via Trapping of the Glycosyl-Enzyme Intermediate
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- 作者：Curtis Braun ; Thisbe Lindhorst ; Neil B. Madsen ; and Stephen G. Withers
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：April 30, 1996
- 年：1996
- 卷：35
- 期：17
- 页码：5458 - 5463
- 全文大小：644K
- 年卷期：v.35,no.17(April 30, 1996)
- ISSN：1520-4995
文摘
Glycogen-debranching enzyme catalyzes the removal of branchingfrom glycogen via a two-step process involving first the transfer of a maltotriosyl unit fromthe branch to the main chain andsecond the hydrolysis of the residual 
-(1,6)-linked glucose moiety.Since the transfer occurs with retentionof anomeric configuration, a mechanism involving amaltotriosyl-enzyme species is presumed. 4-Deoxy--maltotriosyl fluoride functions as an incompetent substrate for thistransferase activity since a glycosyl-enzyme species is formed, as witnessed by a "burst" of fluoriderelease, but turned over only very slowlyunless a suitable acceptor such as maltotriose is added, at which point4-deoxymaltohexaose is released.Peptic proteolysis of this trapped enzyme generated a mixture ofpeptides which was separated by reversephase high-performance liquid chromatography, and the glycosylatedpeptide was located by use of tandemmass spectrometry in the neutral loss mode. Subsequent tandem massspectrometric experiments on thispeptide identified it as one surrounding Asp 549. This amino acidis completely conserved in all-glucanotransferases and -glucosidases belonging to thissequence-related family and is hereby identifiedas the catalytic nucleophile.
-(1,6)-linked glucose moiety.Since the transfer occurs with retentionof anomeric configuration, a mechanism involving amaltotriosyl-enzyme species is presumed. 4-Deoxy--maltotriosyl fluoride functions as an incompetent substrate for thistransferase activity since a glycosyl-enzyme species is formed, as witnessed by a "burst" of fluoriderelease, but turned over only very slowlyunless a suitable acceptor such as maltotriose is added, at which point4-deoxymaltohexaose is released.Peptic proteolysis of this trapped enzyme generated a mixture ofpeptides which was separated by reversephase high-performance liquid chromatography, and the glycosylatedpeptide was located by use of tandemmass spectrometry in the neutral loss mode. Subsequent tandem massspectrometric experiments on thispeptide identified it as one surrounding Asp 549. This amino acidis completely conserved in all-glucanotransferases and -glucosidases belonging to thissequence-related family and is hereby identifiedas the catalytic nucleophile.