PMID:7829480
| Citation |
Frick, DN and Bessman, MJ (1995) Cloning, purification, and properties of a novel NADH pyrophosphatase. Evidence for a nucleotide pyrophosphatase catalytic domain in MutT-like enzymes. J. Biol. Chem. 270:1529-34 |
|---|---|
| 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 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. |
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| Keywords |
Amino Acid Sequence; Bacterial Proteins/chemistry; Bacterial Proteins/metabolism; Binding Sites; Cations, Divalent; Chlorides/pharmacology; Chromatography, Gel; Chromatography, Ion Exchange; Cloning, Molecular; DNA Primers; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins; Kinetics; Magnesium Chloride/pharmacology; Manganese Compounds/pharmacology; Molecular Sequence Data; Molecular Weight; Open Reading Frames; Phosphoric Monoester Hydrolases/chemistry; Phosphoric Monoester Hydrolases/metabolism; Pyrophosphatases/biosynthesis; Pyrophosphatases/isolation & purification; Pyrophosphatases/metabolism; Recombinant Proteins/biosynthesis; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Sequence Homology, Amino Acid; Substrate Specificity |
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