Leech A P, Boetzel R, McDonald C, Shrive A K, Moore G R, Coggins J R, Sawyer L, Kleanthous C
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom.
J Biol Chem. 1998 Apr 17;273(16):9602-7. doi: 10.1074/jbc.273.16.9602.
Type I dehydroquinase from the shikimate pathway of Escherichia coli dehydrates dehydroquinate to dehydroshikimate. pH/log Vmax profiles of the enzyme indicate the presence of a single ionizing group with a pKa of 6.2. Chemical modification experiments with diethyl pyrocarbonate have identified the conserved residue His-143 as essential for catalysis in this enzyme and the pKa for this modification is also 6.2, implying that this is the single ionizing residue in dehydroquinase that may be acting as a general base in the catalytic mechanism. Subsequent mutagenesis of this residue (Leech, A. P., James, R., Coggins, J. R., and Kleanthous, C. (1995) J. Biol. Chem. 270, 25827-25836) further suggested that His-143 may be involved in Schiff base formation/breakdown as well as being the proton abstracting general base. The importance of this residue was confirmed by recent x-ray crystallographic data showing His-143 to be at the center of a hydrogen-bonded triad, flanked by the essential Schiff base forming residue Lys-170 and Glu-86. In the present study, we have used mutagenesis and 1H and 13C NMR to assign the resonance of His-143 and probe its ionization state to define more precisely its role in the mechanism of type I dehydroquinase. Following isotopic enrichment of wild-type and H143A dehydroquinase enzymes with [2-13C]histidine, the resonance for His-143 was assigned by comparing their 1H,13C heteronuclear single quantum correlation NMR spectra. pH titrations revealed that whether in the liganded or unliganded state, His-143 does not ionize over the pH range 6-9.5 and so cannot possess a pKa of 6.2. The NMR data are consistent with this residue remaining unprotonated at pH values optimal for the activity of this enzyme (pH > 7). The role of His-143 is re-evaluated in light of these and the recent structural data, and an alternative candidate for the pKa of 6.2 is discussed.
来自大肠杆菌莽草酸途径的I型脱氢奎尼酸酶将脱氢奎尼酸脱水生成脱氢莽草酸。该酶的pH/最大反应速度对数曲线表明存在一个单一的电离基团，其pKa为6.2。用焦碳酸二乙酯进行的化学修饰实验已确定保守残基His-143是该酶催化作用所必需的，且此修饰的pKa也是6.2，这意味着这是脱氢奎尼酸酶中可能在催化机制中充当通用碱的单一电离残基。随后对该残基进行的诱变（Leech, A. P., James, R., Coggins, J. R., and Kleanthous, C. (1995) J. Biol. Chem. 270, 25827 - 25836）进一步表明His-143可能参与席夫碱的形成/分解以及作为夺取质子的通用碱。最近的X射线晶体学数据证实了该残基的重要性，数据显示His-143位于一个氢键三联体的中心，两侧是形成席夫碱所必需的残基Lys-170和Glu-86。在本研究中，我们利用诱变以及1H和13C核磁共振来确定His-143的共振信号并探测其电离状态，以更精确地界定其在I型脱氢奎尼酸酶机制中的作用。在用[2-13C]组氨酸对野生型和H143A脱氢奎尼酸酶进行同位素富集后，通过比较它们的1H,13C异核单量子相关核磁共振谱来确定His-143的共振信号。pH滴定显示，无论处于结合态还是未结合态，His-143在pH值6 - 9.5范围内都不会电离，因此不可能具有6.2的pKa。核磁共振数据与该残基在该酶活性最佳的pH值（pH > 7）下保持未质子化状态一致。根据这些以及最近的结构数据重新评估了His-143的作用，并讨论了pKa为6.2的另一个候选残基。
