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首页> 外文期刊>Biochemistry >Site-Directed mutagenesis of human nucleside triphosphate diphosphate diphosphohydrolase 3:the importance of residues i nthe apyrase conserved regions
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Site-Directed mutagenesis of human nucleside triphosphate diphosphate diphosphohydrolase 3:the importance of residues i nthe apyrase conserved regions

机译：人核苷三磷酸二磷酸二磷酸二氢水解酶的定点诱变3：腺苷三磷酸双磷酸酶保守区残基的重要性

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摘要

Ecto-nucleoside triphosphate diphosphohydrolase 3 (eNTPDase-3, also known as HB6 and CD39L3) is a membrane-associated ecto-apyrase. Only a few functionally significant residues have been elucidated for this enzyme, as well as for the whole family of eNTPDase enzymes. Four highly conserved regions (apyrase conserved regions, ACRs) have been identified in all the members of eNTPDase family, suggesting their importance for biological activity. In an effort to identify those amino acids important for the catalytic activity of the eNTPDase family, as well as those residues mediating substrate specificity, 11 point mutations of 7 amino acid residues in ACRI —4 of eNTPDase-3 were constructed by site-directed mutagenesis. Mutagenesis of asparagine 191 to alanine (N191A), glutamine 226 to alanine (Q226A), and arginine 67 to glycine (R67G) resulted in an increase in the rates of hydrolysis of nucleoside diphosphates relative to triphosphates. Mutagenesis of arginine 146 to proline (R146P) essentially converted the eNTPDase-3 ecto-apyrase to an ecto-ATPase (eNTPDase-2), mainly by decreasing the hydrolysis rates for nucleoside diphosphates. The Q226A mutant exhibited a change in the divalent cation requirement for nucleotidase activity relative to the wild-type and the other mutants. Mutation of glutamate 182 to aspartate (El 82D) or glutamine (El 82Q), and mutation of serine 224 to alanine (S224A) completely abolished enzymatic activity. We conclude that the residues corresponding to eNTPDase-3 glutamate 182 in ACR3 and serine 224 in ACR4 are essential for the enzymatic activity of eNTPDases in general, and that arginine 67, arginine 146, asparagine 191, and glutamine 226 are important for determining substrate specificity for human ecto-nucleoside triphosphate diphosphohydrolase 3.
机译：胞外核苷三磷酸二磷酸水解酶3（eNTPDase-3，也称为HB6和CD39L3）是一种与膜相关的胞外Apyrase。对于该酶以及整个eNTPDase酶家族，仅阐明了几个功能上重要的残基。在eNTPDase家族的所有成员中已经鉴定出四个高度保守的区域（apyrase保守区域，ACR），表明它们对生物学活性的重要性。为了鉴定对eNTPDase家族的催化活性重要的氨基酸以及介导底物特异性的那些残基，通过定点诱变构建了eNTPDase-3的ACRI -4中7个氨基酸残基的11个点突变。 。天冬酰胺191转化为丙氨酸（N191A），谷氨酰胺226转化为丙氨酸（Q226A），精氨酸67转化为甘氨酸（R67G）的诱变导致相对于三磷酸酯而言，核苷二磷酸酯的水解速率增加。精氨酸146向脯氨酸的诱变（R146P）主要通过降低二磷酸核苷的水解速率将eNTPDase-3胞外磷酸酶转化为ecto-ATPase（eNTPDase-2）。相对于野生型和其他突变体，Q226A突变体在核苷酸酶活性的二价阳离子需求方面表现出变化。谷氨酸182突变为天冬氨酸（E1 82D）或谷氨酰胺（E1 82Q），以及丝氨酸224突变为丙氨酸（S224A）完全消除了酶活性。我们得出的结论是，与ACR3中的eNTPDase-3谷氨酸182和ACR4中的丝氨酸224相对应的残基通常对eNTPDase的酶活性至关重要，精氨酸67，精氨酸146，天冬酰胺191和谷氨酰胺226对于确定底物特异性很重要用于人类外核苷三磷酸二磷酸水解酶3。

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