James Peter L, Anthony Christopher
Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Southampton S016 7PX, Hants, UK.
Biochim Biophys Acta. 2003 Apr 11;1647(1-2):200-5. doi: 10.1016/s1570-9639(03)00041-4.
All pyrroloquinoline quinone (PQQ)-containing dehydrogenases whose structures are known contain Ca(2+) bonded to the PQQ at the active site. However, membrane glucose dehydrogenase (GDH) requires reconstitution with PQQ and Mg(2+) ions (but not Ca(2+)) for activity. To address the question of whether the Mg(2+) replaces the usual active site Ca(2+) in this enzyme, mutant GDHs were produced in which residues proposed to be involved in binding metal ion were modified (D354N-GDH and N355D-GDH and D354N-GDH/N355D-GDH). The most remarkable observation was that reconstitution with PQQ of the mutant enzymes was not supported by Mg(2+) ions as in the wild-type GDH, but it could be supported by Ca(2+), Sr(2+) or Ba(2+) ions. This was competitively inhibited by Mg(2+). This result, together with studies on the kinetics of the modified enzymes have led to the conclusion that, although a Ca(2+) ion is able to form part of the active site of the genetically modified GDH, as in all other PQQ-containing quinoproteins, a Mg(2+) ion surprisingly replaces Ca(2+) in the active site of the wild-type GDH.
所有已知结构的含吡咯喹啉醌（PQQ）的脱氢酶在活性位点都含有与PQQ结合的Ca(2+)。然而，膜葡萄糖脱氢酶（GDH）需要用PQQ和Mg(2+)离子（而非Ca(2+)）进行重组才能具有活性。为了解决Mg(2+)是否在该酶中取代了通常的活性位点Ca(2+)这一问题，制备了突变型GDH，其中对推测参与结合金属离子的残基进行了修饰（D354N-GDH、N355D-GDH和D354N-GDH/N355D-GDH）。最显著的观察结果是，与野生型GDH不同，突变酶与PQQ的重组不受Mg(2+)离子支持，但可受Ca(2+)、Sr(2+)或Ba(2+)离子支持。这受到Mg(2+)的竞争性抑制。这一结果，连同对修饰酶动力学的研究，得出的结论是，尽管与所有其他含PQQ的醌蛋白一样，Ca(2+)离子能够成为基因修饰的GDH活性位点的一部分，但令人惊讶的是，在野生型GDH的活性位点中Mg(2+)取代了Ca(2+)。
