Single-turnover analysis of mutant human apurinic/apyrimidinic endonuclease.

Apurinic/apyrimidinic endonuclease (AP endo) is a key enzyme in the repair of oxidatively damaged DNA. Using single-turnover conditions, we recently described substrate binding parameters for wild type human AP endo. In this study, we utilized four enzyme mutants, D283A, D308A, D283A/D308A, and H309N, and assayed them under steady state and single-turnover conditions. The turnover number of the single aspartate mutants was decreased 10-30-fold in comparison to that of the wild type. The decrease in the turnover number was accompanied by a 17- and 50-fold decrease in the forward rate constant (kon) for substrate binding by D308A and D283A, respectively. The dissociation rate constant for substrate (koff) was unchanged for the D308A mutant but was 10 times faster for the D283A mutant than for the wild type. The apparent Km values for both of the single aspartate mutants were about equal to their respective KD values. To account for the kinetic behavior of the D308A mutant, it was necessary to insert a conformational change into the kinetic scheme. In contrast to the single aspartate mutants, the turnover number for the double mutant was 500-fold lower than that of the wild type, its apparent Km was 2.5-fold higher, and binding to substrate was weak. Mutation of His309 caused the greatest decrease in activity, resulting in a turnover number that was more than 30000-fold lower than that of the wild type and an apparent Km that was 13-fold higher, supporting the notion that His309 is intimately involved in catalysis. Molecular dynamics simulation techniques suggested that conversion of either aspartate to alanine resulted in major shifts in the spatial localization of key amino acids. Despite the fact that the two aspartates flank His309, the movement they engendered was distinct, consistent with the differences in catalytic behavior. We suggest that the conformation of the active site is largely maintained by the two aspartates, which enable efficient binding and cleavage of abasic site-containing DNA.