Role of Arg211 in the Active Site of Human 
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- 作者：Yongmin Hou ; David Vocadlo ; Stephen Withers ; and Don Mahuran
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：May 23, 2000
- 年：2000
- 卷：39
- 期：20
- 页码：6219 - 6227
- 全文大小：153K
- 年卷期：v.39,no.20(May 23, 2000)
- ISSN：1520-4995
文摘
Tay-Sachs or Sandhoff disease results from a deficiency of either the 
- or the 
-subunits of-hexosaminidase A, respectively. These evolutionarily related subunits have been grouped with the "Family20" glycosidases. Molecular modeling of human hexosaminidase has been carried out on the basis of thethree-dimensional structure of a bacterial member of Family 20, Serratia marcescens chitobiase. Theprimary sequence identity between the two enzymes is only 26% and restricted to their active site regions;therefore, the validity of this model must be determined experimentally. Because human hexosaminidasecannot be functionally expressed in bacteria, characterization of mutagenized hexosaminidase must becarried out using eukaryotic cell expression systems that all produce endogenous hexosaminidase activity.Even small amounts of endogenous enzyme can interfere with accurate Km or Vmax determinations. Wereport the expression, purification, and characterization of a C-terminal His6-tag precursor form of hexosaminidase B that is 99.99% free of endogenous enzyme from the host cells. Control experiments arereported confirming that the kinetic parameters of the His6-tag precursor are the same as the untaggedprecursor, which in turn are identical to the mature isoenzyme. Using highly purified wild-type and Arg211Lys-substituted hexosaminidase B, we reexamine the role of Arg211 in the active site. As we previouslyreported, this very conservative substitution nevertheless reduces kcat by 500-fold. However, the removalof all endogenous activity has now allowed us to detect a 10-fold increase in Km that was not apparent inour previous study. That this increase in Km reflects a decrease in the strength of substrate binding wasconfirmed by the inability of the mutant isozyme to efficiently bind an immobilized substrate analogue,i.e., a hexosaminidase affinity column. Thus, Arg211 is involved in substrate binding, as predicted by thechitobiase model, as well as catalysis.
- or the -subunits of-hexosaminidase A, respectively. These evolutionarily related subunits have been grouped with the "Family20" glycosidases. Molecular modeling of human hexosaminidase has been carried out on the basis of thethree-dimensional structure of a bacterial member of Family 20, Serratia marcescens chitobiase. Theprimary sequence identity between the two enzymes is only 26% and restricted to their active site regions;therefore, the validity of this model must be determined experimentally. Because human hexosaminidasecannot be functionally expressed in bacteria, characterization of mutagenized hexosaminidase must becarried out using eukaryotic cell expression systems that all produce endogenous hexosaminidase activity.Even small amounts of endogenous enzyme can interfere with accurate Km or Vmax determinations. Wereport the expression, purification, and characterization of a C-terminal His6-tag precursor form of hexosaminidase B that is 99.99% free of endogenous enzyme from the host cells. Control experiments arereported confirming that the kinetic parameters of the His6-tag precursor are the same as the untaggedprecursor, which in turn are identical to the mature isoenzyme. Using highly purified wild-type and Arg211Lys-substituted hexosaminidase B, we reexamine the role of Arg211 in the active site. As we previouslyreported, this very conservative substitution nevertheless reduces kcat by 500-fold. However, the removalof all endogenous activity has now allowed us to detect a 10-fold increase in Km that was not apparent inour previous study. That this increase in Km reflects a decrease in the strength of substrate binding wasconfirmed by the inability of the mutant isozyme to efficiently bind an immobilized substrate analogue,i.e., a hexosaminidase affinity column. Thus, Arg211 is involved in substrate binding, as predicted by thechitobiase model, as well as catalysis.