Structure and Mechanism of Action of an Inverting Mutant Sialidase
详细信息 查看全文
- 作者：Simon Newstead ; Jacqueline N. Watson ; Tara L. Knoll ; Andrew J. Bennet ; and Garry Taylor
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：June 28, 2005
- 年：2005
- 卷：44
- 期：25
- 页码：9117 - 9122
- 全文大小：295K
- 年卷期：v.44,no.25(June 28, 2005)
- ISSN：1520-4995
文摘
Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidaseto small amino acids changes the mechanism of catalysis from retention of anomeric configuration toinversion [Watson, J. N., et al. (2003) Biochemistry 42, 12682-12690]. For the Y370G mutant enzyme-catalyzed hydrolysis of a series of aryl sialosides and 3'-sialyllactose, the derived Br
nsted parameters(
lg) on kcat and kcat/Km are -0.63 ± 0.05 and -0.80 ± 0.08, respectively. Thus, for the Y370G enzyme,glycosidic C-O bond cleavage is rate-determining. Analysis of the activity of the Y370G mutant andwild-type enzymes against a substrate [3,4-dihydro-2H-pyrano[3,2-c]pyridinium 
-D-N-acetylneuraminide(DHP-Neu5Ac)] whose hydrolysis cannot be accelerated by acid catalysis is consistent with these reactionsproceeding via SN1 and SN2 mechanisms, respectively. The overall structure of the Y370G mutant sialidaseactive site is very similar to the previously reported wild-type structure [Gaskell, A., et al. (1995) Structure3, 1197-1205], although removal of the tyrosine residue creates two significant changes to the activesite. First, the anomeric oxygen atom of the hydrolysis product (-N-acetylneuraminic acid) and fourwater molecules bind in the large cavity created by the Y370G mutation. Second, the side chain of Asn310moves to make a strong hydrogen bond to one of the bound water molecules.
nsted parameters(lg) on kcat and kcat/Km are -0.63 ± 0.05 and -0.80 ± 0.08, respectively. Thus, for the Y370G enzyme,glycosidic C-O bond cleavage is rate-determining. Analysis of the activity of the Y370G mutant andwild-type enzymes against a substrate [3,4-dihydro-2H-pyrano[3,2-c]pyridinium -D-N-acetylneuraminide(DHP-Neu5Ac)] whose hydrolysis cannot be accelerated by acid catalysis is consistent with these reactionsproceeding via SN1 and SN2 mechanisms, respectively. The overall structure of the Y370G mutant sialidaseactive site is very similar to the previously reported wild-type structure [Gaskell, A., et al. (1995) Structure3, 1197-1205], although removal of the tyrosine residue creates two significant changes to the activesite. First, the anomeric oxygen atom of the hydrolysis product (-N-acetylneuraminic acid) and fourwater molecules bind in the large cavity created by the Y370G mutation. Second, the side chain of Asn310moves to make a strong hydrogen bond to one of the bound water molecules.