Identification of potential active-site residues in the human poly(ADP-ribose) polymerase.
The carboxyl-terminal catalytic domain of the human poly(ADP-ribose) polymerase (PARP) exhibits sequence homology with the NAD(P)(+)-dependent leucine and glutamate dehydrogenases. To clarify the role played by some conserved residues between PARP and NAD(P)(+)-dependent dehydrogenases, point mutations were introduced into the whole enzyme context. Non-conservative mutations of Lys-893 (K893I) and Asp-993 (D993A) completely inactivate human PARP, whereas conservative and nonconservative mutations of Asp-914 (D914E and D914A, respectively) and Lys-953 (K953R and K953I, respectively) partially alter PARP activity. The consequences of conservative substitution of Lys-893 and Asp-993 on the kinetic properties of human poly(ADP-ribose) polymerase enzyme and the polymer it synthesizes suggest that these 2 amino acids are directly involved in the covalent attachment of the first ADP-ribosyl residue from NAD+ onto the acceptor amino acid. In addition, the recent resolution of the three-dimensional structure of the NAD(+)-linked glutamate dehydrogenase from Clostridium symbiosum (Baker, P.J., Britton, K.L., Engel, P.C., Farrants, G.W., Lilley, K.S., Rice, D.W., and Stillman, T.J. (1992) Proteins 12, 75-86) strongly supports our alignment with leucine and glutamate dehydrogenases and provides an interesting structural framework for the analysis of our results of site-directed mutagenesis.
Simonin
F
F
Unité propre de recherche de Cancérogenèse et de Mutagenèse Moléculaire et Structurale, Centre National de la Recherche Scientifique, Strasbourg, France.
Poch
O
O
Delarue
M
M
de Murcia
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
D011065:Poly(ADP-ribose) Polymerases