Frick D N, Bessman M J
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
J Biol Chem. 1995 Jan 27;270(4):1529-34. doi: 10.1074/jbc.270.4.1529.
An Escherichia coli open reading frame containing significant homology to the active site of the MutT enzyme codes for a novel dinucleotide pyrophosphatase. The motif shared by these two proteins and several others is conserved throughout nature and may designate a nucleotide-binding or pyrophosphatase domain. The E. coli NADH pyrophosphatase has been cloned, overexpressed, and purified to near homogeneity. The protein contains 257 amino acids (M(r) = 29,774) and migrates on gel filtration columns as an apparent dimer. The enzyme catalyzes the hydrolysis of a broad range of dinucleotide pyrophosphates, but uniquely prefers the reduced form of NADH. The Vmax/Km for NADH (69 mumol min-1 mg-1 mM-1) is an order of magnitude higher than for any other dinucleotide pyrophosphate tested. In addition, the Km for NADH (0.1 mM) is 50-fold lower than the Km for NAD+. The hydrolysis of dinucleotide pyrophosphates requires divalent metal ions and yields two mononucleoside 5'-phosphates. The metals that most efficiently stimulate activity are Mg2+ and Mn2+. Although these metals support similar Vmax values at optimal metal concentration, the apparent Km for Mg2+ is 3.7 mM (at 1 mM NADH), whereas the apparent Km for Mn2+ at the same NADH concentration is 30 microM.
一个与MutT酶活性位点具有显著同源性的大肠杆菌开放阅读框编码一种新型二核苷酸焦磷酸酶。这两种蛋白质以及其他几种蛋白质共有的基序在自然界中是保守的，可能代表一个核苷酸结合或焦磷酸酶结构域。大肠杆菌NADH焦磷酸酶已被克隆、过量表达并纯化至接近均一。该蛋白质含有257个氨基酸（相对分子质量=29774），在凝胶过滤柱上以明显的二聚体形式迁移。该酶催化多种二核苷酸焦磷酸的水解，但独特地偏好还原形式的NADH。NADH的Vmax/Km（69μmol min-1 mg-1 mM-1）比测试的任何其他二核苷酸焦磷酸高一个数量级。此外，NADH的Km（0.1 mM）比NAD+的Km低50倍。二核苷酸焦磷酸的水解需要二价金属离子，并产生两个5'-单磷酸核苷。最有效地刺激活性的金属是Mg2+和Mn2+。尽管这些金属在最佳金属浓度下支持相似的Vmax值，但Mg2+的表观Km为3.7 mM（在1 mM NADH时），而在相同NADH浓度下Mn2+的表观Km为30μM。
