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 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.
Frick
D N
DN
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Bessman
M J
MJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002413:Cations, Divalent; D002712:Chlorides / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D017895:Manganese Compounds / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D011755:Pyrophosphatases / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002413:Cations, Divalent; D002712:Chlorides; D017931:DNA Primers; D029968:Escherichia coli Proteins; D017895:Manganese Compounds; D011994:Recombinant Proteins; D015636:Magnesium Chloride; D010744:Phosphoric Monoester Hydrolases; D011755:Pyrophosphatases; C052211:mutT protein, E coli; C021899:nucleotide pyrophosphatase; C025340:manganese chloride