Presteady-state analysis of a single catalytic turnover by Escherichia coli uracil-DNA glycosylase reveals a "pinch-pull-push" mechanism.
Uracil-DNA glycosylase catalyzes the excision of uracils from DNA via a mechanism where the uracil is extrahelically flipped out of the DNA helix into the enzyme active site. A conserved leucine is inserted into the DNA duplex space vacated by the uracil leading to the paradigmatic "push-pull" mechanism of nucleotide flipping. However, the order of these two steps during catalysis has not been conclusively established. We report a complete kinetic analysis of a single catalytic turnover using a hydrolyzable duplex oligodeoxyribonucleotide substrate containing a uracil:2-aminopurine base pair. Rapid chemical-quenched-flow methods defined the kinetics of excision at the active site during catalysis. Stopped-flow fluorometry monitoring the 2-aminopurine fluorescence defined the kinetics of uracil flipping. Parallel experiments detecting the protein fluorescence showed a slower Leu(191) insertion step occurring after nucleotide flipping but before excision. The inserted Leu(191) acts as a doorstop to prevent the return of the flipped-out uracil residue, thereby facilitating the capture of the uracil in the active site and does not play a direct role in "pushing" the uracil out of the DNA helix. The results define for the first time the proper sequence of events during a catalytic cycle and establish a "pull-push", as opposed to a "push-pull", mechanism for nucleotide flipping.
Wong
Isaac
I
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, USA. wongis@onid.orst.edu
Lundquist
Amy J
AJ
Bernards
Andrew S
AS
Mosbaugh
Dale W
DW
D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D045647:DNA Glycosylases; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014498:Uracil / Q000737:chemistry; D051981:Uracil-DNA Glycosidase
D009838:Oligodeoxyribonucleotides; D014498:Uracil; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D007930:Leucine