Identifying the Catalytic Acid/Base in GH29 伪-l-Fucosidase Subfamilies
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- 作者：F. Aidha Shaikh ; Alicia Lammerts van Bueren ; Gideon J. Davies ; Stephen G. Withers
- 刊名：Biochemistry
- 出版年：2013
- 出版时间：August 27, 2013
- 年：2013
- 卷：52
- 期：34
- 页码：5857-5864
- 全文大小：343K
- 年卷期：v.52,no.34(August 27, 2013)
- ISSN：1520-4995
文摘
While the catalytic nucleophile in the configuration-retaining 伪-l-fucosidases from family GH29 is fully conserved with respect to sequence, there is no fully sequence-conserved acid/base residue candidate across the family. X-ray crystallographic studies and kinetic characterizations have allowed the identification of this residue in a few cases, and a recent combination of phylogenetic tree analyses with substrate specificity data has allowed the division of GH29 enzymes into two subfamilies, A and B, allowing the probable assignment of these residues. Here, we perform detailed kinetic and mechanistic characterizations of the corresponding alanine mutants and other candidates. Through comparison of kinetic parameters obtained for the hydrolysis of fucosyl substrates with activated leaving groups by these mutants with those of the corresponding wild-type enzymes, in conjunction with the demonstration of azide rescue, we largely confirm the acid/base residue predictions for the GH29 fucosidases from the two subfamilies.