CLONING, PURIFICATION, AND PROPERTIES OF A NOVEL NADH PYROPHOSPHATASE - EVIDENCE FOR A NUCLEOTIDE PYROPHOSPHATASE CATALYTIC DOMAIN IN MUTT-LIKE ENZYMES

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-m for NADH (69 mu mol min(-1) mg(-1) mM(-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 mu M.