The role of histidine 632 in catalysis by human topoisomerase I

Based on the crystal structure of human topoisomerase I, we hypothesized th at hydrogen bonding between the side chain of the highly conserved His(632) and one of the nonbridging oxygens of the scissile phosphate contributes t o catalysis by stabilizing the transition state. This hypothesis has been t ested by examining the effects of changing His(632) to glutamine, asparagin e, alanine, and tryptophan, The change to glutamine reduced both the relaxa tion activity and single-turnover cleavage activity by approximately 100-fo ld, whereas the same change at three other conserved histidines (positions 222, 387, and 406) had no significant effect on the relaxation activity. Th e properties of the mutant protein containing asparagine instead of histidi ne at position 632 were similar to those of the glutamine mutant, whereas m utations to alanine or tryptophan reduced the activity by approximately 4 o rders of magnitude. The reduction in activity for the mutants was not due t o alterations in substrate binding affinities or changes in the cleavage sp ecificities of the proteins. The above results for the glutamine mutation i n conjunction with the similar effects of pH on the wild type and the H632Q mutant enzyme rule out the possibility that His(632) acts as a general aci d to protonate the leaving 5'-oxygen during the cleavage reaction, Taken to gether, these data strongly support the hypothesis that the only role for H is632 is to stabilize the pentavalent transition state through hydrogen bon ding to one of the nonbridging oxygens.