The carboxyl terminus of coffee bean α-galactosidase is critical for enzyme activity

被引:9
|
作者
Maranville, E [1 ]
Zhu, A [1 ]
机构
[1] New York Blood Ctr, Lindsley F Kimball Res Inst, New York, NY 10021 USA
关键词
alpha-galactosidase; coffee; mutagenesis; enzymatic activity; Pichia pastoris;
D O I
10.1006/abbi.1999.1532
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
The role of the carboxyl (C)-terminal region of coffee bean alpha-galactosidase (alpha-GAL) has been studied by expressing C-terminal deletion mutants in the methylotrophic yeast strain Pichia pastoris. A previous study of human ly-galactosidase determined that enzyme activity increased when up to 10 amino acid residues were deleted. Deleting 11 residues reduced activity, and deleting 12 residues abolished activity. In our studies, alpha-GAL activity is reduced when one or two amino acids are deleted, as is enzyme secretion directed by P, pastoris signal sequences. The pH profile is similar to that of the wild-type enzyme. Deleting 3 or more residues from the C-terminal end results in a complete loss of both enzyme secretion and activity. The C-terminus of alpha-GAL seems to play an important role in overall enzyme conformation and may directly affect the proper conformation of the active site. (C) 2000 Academic Press.
引用
收藏
页码:225 / 230
页数:6
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