Role of βArg211 in the active site of human β-hexosaminidase B

被引:13
作者
Hou, YM
Vocadlo, D
Withers, S
Mahuran, D
机构
[1] Hosp Sick Children, Res Inst, Toronto, ON M5G 1X8, Canada
[2] Dept Lab Med & Pathobiol, Toronto, ON M5G 2C4, Canada
[3] Univ British Columbia, Dept Chem, Vancouver, BC V6T 1Z1, Canada
基金
加拿大健康研究院;
关键词
D O I
10.1021/bi992464j
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Tay-Sachs or Sandhoff disease results from a deficiency of either the alpha- or the beta-subunits of beta-hexosaminidase A, respectively. These evolutionarily related subunits have been grouped with the "Family 20" glycosidases. Molecular modeling of human hexosaminidase has been carried out on the basis of the three-dimensional structure of a bacterial member of Family 20, Serratia marcescens chitobiase. The primary 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 hexosaminidase cannot be functionally expressed in bacteria, characterization of mutagenized hexosaminidase must be carried out using eukaryotic cell expression systems that all produce endogenous hexosaminidase activity. Even small amounts of endogenous enzyme can interfere with accurate K-m or V-max determinations. We report the expression, purification, and characterization of a C-terminal Hiss-tag precursor form of hexosaminidase B that is 99.99% free of endogenous enzyme from the host cells. Control experiments are reported confirming that the kinetic parameters of the His(6)-tag precursor are the same as the untagged precursor, which in turn are identical to the mature isoenzyme. Using highly purified wild-type and Arg(211)- Lys-substituted hexosaminidase B, we reexamine the role of Arg(211) in the active site. As we previously reported, this very conservative substitution nevertheless reduces k(cat) by 500-fold. However, the removal of all endogenous activity has now allowed us to detect a 10-fold increase in K-m that was not apparent in our previous study. That this increase in K-m reflects a decrease in the strength of substrate binding was confirmed by the inability of the mutant isozyme to efficiently bind an immobilized substrate analogue, i.e., a hexosaminidase affinity column. Thus, Arg(211) is involved in substrate binding, as predicted by the chitobiase model, as well as catalysis.
引用
收藏
页码:6219 / 6227
页数:9
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