title: Analysis of human tyrosyl-DNA phosphodiesterase I catalytic residues.
abstract: Tyrosyl-DNA phosphodiesterase I (Tdp1) is involved in the repair of DNA lesions created by topoisomerase I in vivo. Tdp1 is a member of the phospholipase D (PLD) superfamily of enzymes and hydrolyzes 3'-phosphotyrosyl bonds to generate 3'-phosphate DNA and free tyrosine in vitro. Here, we use synthetic 3'-(4-nitro)phenyl, 3'-(4-methyl)phenyl, and 3'-tyrosine phosphate oligonucleotides to study human Tdp1. Kinetic analysis of human Tdp1 (hTdp1) shows that the enzyme has nanomolar affinity for all three substrates and the overall in vitro reaction is diffusion-limited. Analysis of active-site mutants using these modified substrates demonstrates that hTdp1 uses an acid/base catalytic mechanism. The results show that histidine 493 serves as the general acid during the initial transesterification, in agreement with hypotheses based on previous crystal structure models. The results also argue that lysine 495 and asparagine 516 participate in the general acid reaction, and the analysis of crystal structures suggests that these residues may function in a proton relay. Together with previous crystal structure data, the new functional data provide a mechanistic understanding of the conserved histidine, lysine and asparagine residues found among all PLD family members.
authors.affiliation: Biology Department, San Diego State University, CA 98182-4614, USA.
authors.forename: Amy C
authors.initials: AC
authors.lastname: Raymond
authors.forename: Marc C
authors.initials: MC
authors.lastname: Rideout
authors.forename: Bart
authors.initials: B
authors.lastname: Staker
authors.forename: Kathryn
authors.initials: K
authors.lastname: Hjerrild
authors.forename: Alex B
authors.initials: AB
authors.lastname: Burgin
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D010727:Phosphoric Diester Hydrolases; C498508:TDP1 protein, human; C122147:tyrosyl-DNA phosphodiesterase; D004264:DNA Topoisomerases, Type I