Libessart N, Preiss J
Department of Biochemistry, Michigan State University, East Lansing, Michigan, 48824, USA.
Arch Biochem Biophys. 1998 Dec 1;360(1):135-41. doi: 10.1006/abbi.1998.0960.
Branching enzyme (BE) belongs to the amylolytic family which contains four highly conserved regions. These regions are proposed to play an important role in catalysis as they are thought to be necessary for catalysis and/or binding the substrate. Only one arginine residue was found to be conserved in a catalytic center at the same position in all known sequences of BEs from various species as well as in the alpha-amylase enzyme family. In mBEII, a conserved Arg residue 384 is in catalytic region 2. We have used site-directed mutagenesis of the Arg-384 residue in order to study its possible role in BE. Previous chemical modification studies (H. Cao and J. Preiss, 1996, J. Prot. Chem. 15, 291-304) suggest that it may play a role in substrate binding. Replacement of Arg-384 by Ala, Ser, Gln, and Glu in the active site caused almost total inactivation. However, a conservative mutation of the conserved Arg-384 by Lys resulted in some residual activity, approximately 5% of the wild-type enzyme. The kinetics of the purified mutant R384K enzyme were investigated and no large effect on the Km of the substrate amylose for BE was observed. Thus, these results suggest that conserved Arg residue 384 in mBEII plays an important role in the catalytic function of BEs but may not be directly involved in substrate binding.
分支酶（BE）属于淀粉分解酶家族，该家族包含四个高度保守的区域。这些区域被认为在催化过程中起重要作用，因为它们被认为是催化和/或结合底物所必需的。在来自各种物种的BE的所有已知序列以及α-淀粉酶家族中，仅发现一个精氨酸残基在催化中心的相同位置保守。在mBEII中，保守的精氨酸残基384位于催化区域2。我们使用定点诱变精氨酸-384残基来研究其在BE中的可能作用。先前的化学修饰研究（H. Cao和J. Preiss，1996，J. Prot. Chem. 15，291-304）表明它可能在底物结合中起作用。在活性位点用丙氨酸、丝氨酸、谷氨酰胺和谷氨酸取代精氨酸-384几乎导致完全失活。然而，将保守的精氨酸-384保守突变为赖氨酸导致了一些残余活性，约为野生型酶的5%。对纯化的突变体R384K酶的动力学进行了研究，未观察到对BE底物直链淀粉的Km有很大影响。因此，这些结果表明mBEII中保守的精氨酸残基384在BE的催化功能中起重要作用，但可能不直接参与底物结合。
