Binderup K, Preiss J
Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
Biochemistry. 1998 Jun 23;37(25):9033-7. doi: 10.1021/bi980199g.
The branching enzyme belongs to the amylolytic family, a group of enzymes that cleave and/or transfer chains of glucan. The amylolytic enzymes are homologous and all contain four conserved regions, proposed to contain the active site. By primary structure analysis, a conserved position unique to branching enzymes has been identified. This residue, which is either Asp or Glu, depending on the species, is located immediately after the putative catalytic Glu-458 (Escherichia coli numbering). Branching enzymes differ from other amylolytic enzymes in having this acid pair, and we asked if this motif could be essential for branching enzyme action. We used site-directed mutagenesis of the Glu-459 residue in the E. coli branching enzyme in order to determine the significance of the conserved Asp/Glu in branching enzymes. A substitution of Glu-459 to Asp resulted in increased specific activity compared to wild-type, suggesting that the mutation had created a more efficient enzyme. Changing Glu-459 to Ala, Lys, or Gln lowered the specific activities and altered the preferred substrate from amylose to amylopectin.
分支酶属于淀粉分解酶家族，这是一组能够切割和/或转移葡聚糖链的酶。淀粉分解酶具有同源性，并且都包含四个保守区域，推测这些区域含有活性位点。通过一级结构分析，已鉴定出分支酶特有的一个保守位置。该残基根据物种不同，要么是天冬氨酸（Asp），要么是谷氨酸（Glu），位于假定的催化性谷氨酸-458（按照大肠杆菌的编号）之后紧邻的位置。分支酶与其他淀粉分解酶的不同之处在于具有这个酸性氨基酸对，我们探究了这个基序对于分支酶的作用是否至关重要。我们对大肠杆菌分支酶中的谷氨酸-459残基进行了定点诱变，以确定分支酶中保守的天冬氨酸/谷氨酸的重要性。将谷氨酸-459替换成天冬氨酸，与野生型相比，比活性增加，这表明该突变产生了一种更高效的酶。将谷氨酸-459换成丙氨酸、赖氨酸或谷氨酰胺会降低比活性，并使偏好的底物从直链淀粉变为支链淀粉。
