Song Jikui, Bettendorff Lucien, Tonelli Marco, Markley John L
Center for Eukaryotic Structural Genomics and National Magnetic Resonance Facility at Madison, Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706-1544, USA.
J Biol Chem. 2008 Apr 18;283(16):10939-48. doi: 10.1074/jbc.M709675200. Epub 2008 Feb 14.
Mammalian soluble thiamine triphosphatase (ThTPase) is a 25-kDa cytosolic enzyme that specifically catalyzes the conversion of thiamine triphosphate (ThTP) to thiamine diphosphate and has an absolute requirement for divalent cations. We have investigated the kinetic properties of recombinant mouse thiamine triphosphatase (mThTPase) and determined its solution structure by NMR spectroscopy. Residues responsible for binding Mg(2+) and ThTP were determined from NMR titration experiments. The binding of Mg(2+) induced only a minor local conformational change, whereas ThTP binding was found to cause a more global conformational change. We derived a structural model for the mThTPase.ThTP.Mg(2+) ternary complex and concluded from this that whereas free mThTPase has an open cleft fold, the enzyme in the ternary complex adopts a tunnel fold. Our results provide a functional rationale for a number of conserved residues and suggest an essential role for Mg(2+) in catalysis. We propose a mechanism underlying the high substrate specificity of mThTPase and discuss the possible role of water molecules in enzymatic catalysis.
哺乳动物可溶性硫胺素三磷酸酶（ThTPase）是一种25 kDa的胞质酶，它特异性催化硫胺素三磷酸（ThTP）转化为硫胺素二磷酸，并且对二价阳离子有绝对需求。我们研究了重组小鼠硫胺素三磷酸酶（mThTPase）的动力学性质，并通过核磁共振光谱确定了其溶液结构。通过核磁共振滴定实验确定了负责结合Mg(2+)和ThTP的残基。Mg(2+)的结合仅引起轻微的局部构象变化，而ThTP的结合则导致更全局性的构象变化。我们推导了mThTPase.ThTP.Mg(2+)三元复合物的结构模型，并由此得出结论，即游离的mThTPase具有开放裂隙折叠，而三元复合物中的酶则采用隧道折叠。我们的结果为许多保守残基提供了功能依据，并表明Mg(2+)在催化中起重要作用。我们提出了mThTPase高底物特异性的潜在机制，并讨论了水分子在酶催化中的可能作用。
