Cloning, Purification, and Properties of a Novel NADH Pyrophosphatase

Abstract

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 V(max)/K(max) for NADH (69 μmol min-1mg-1mm-1) is an order of magnitude higher than for any other dinucleotide pyrophosphate tested. In addition, the K(m) for NADH (0.1 mM) is 50-fold lower than the K(m) 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 V(max), values at optimal metal concentration, the apparent K(m) for Mg2+is 3.7 mM (at 1 mM NADH), whereas the apparent K(m) for Mn2+at the same NADH concentration is 30 μM.