IDENTIFICATION OF POTENTIAL ACTIVE-SITE RESIDUES IN THE HUMAN POLY(ADP-RIBOSE) POLYMERASE

The carboxyl-terminal catalytic domain of the human poly(ADP-ribose) p olymerase (PARP) exhibits sequence homology with the NAD(P)+-dependent leucine and glutamate dehydrogenases. To clarify the role played by s ome conserved residues between PARP and NAD(P)+-dependent dehydrogenas es, point mutations were introduced into the whole enzyme context. Non -conservative mutations of Lys-893 (K893I) and Asp-993 (D993A) complet ely inactivate human PARP, whereas conservative and nonconservative mu tations of Asp-914 (D914E and D914A, respectively) and Lys-953 (K953R and K953I, respectively) partially alter PARP activity. The consequenc es 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 t he three-dimensional structure of the NAD+-linked glutamate dehydrogen ase 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 leucin e and glutamate dehydrogenases and provides an interesting structural framework for the analysis of our results of site-directed mutagenesis .