Cloning, purification, and properties of a novel NADH pyrophosphatase. Evidence for a nucleotide pyrophosphatase catalytic domain in MutT-like enzymes. J Biol Chem 1995 Jan 27;270(4):1529-34
Date
01/27/1995Pubmed ID
7829480DOI
10.1074/jbc.270.4.1529Scopus ID
2-s2.0-0028900818 (requires institutional sign-in at Scopus site) 89 CitationsAbstract
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.
Author List
Frick DN, Bessman MJAuthor
David N. Frick PhD Associate Professor in the Chimistry & Biochemistry department at University of Wisconsin - MilwaukeeMESH terms used to index this publication - Major topics in bold
Amino Acid SequenceBacterial Proteins
Binding Sites
Cations, Divalent
Chlorides
Chromatography, Gel
Chromatography, Ion Exchange
Cloning, Molecular
DNA Primers
Electrophoresis, Polyacrylamide Gel
Enzyme Activation
Escherichia coli
Escherichia coli Proteins
Kinetics
Magnesium Chloride
Manganese Compounds
Molecular Sequence Data
Molecular Weight
Open Reading Frames
Phosphoric Monoester Hydrolases
Pyrophosphatases
Recombinant Proteins
Sequence Homology, Amino Acid
Substrate Specificity