Uracil DNA glycosylase: revisiting substrate-assisted catalysis by DNA phosphate anions.
Uracil DNA glycosylase (UNG) is a powerful DNA repair enzyme that has been shown to stabilize a glycosyl cation reaction intermediate and a related tight binding inhibitor using electrostatic interactions with the +1 and -1, but not the +2, phosphodiester group of the single-stranded DNA substrate Ap (2+)Ap (1+)Up (1-)ApA. These experimental results differed considerably from computational findings using duplex DNA, where the +2 phosphate was found to stabilize the transition state by approximately 5 kcal/mol, suggesting that UNG uses different catalytic strategies with single-stranded and double-stranded DNA substrates. In addition, the computational studies indicated that the conserved and positively charged His148 (which hydrogen bonds to the +2 phosphate) destabilized the glycosyl cation intermediate by 6-8 kcal/mol through anticatalytic electrostatic interactions. To evaluate these interesting proposals, we measured the kinetic effects of neutral methylphosphonate (MeP) stereoisomers at the +1 and +2 positions of a 12-mer dsDNA substrate and also the catalytic contribution and ionization state of His148. For MeP substitutions at the +1 position, single-turnover kinetic studies showed that the activation barrier was increased by 9.8 and 3.1 kcal/mol, corresponding to a stereoselectivity of nearly 40000-fold for the respective MeP isomers. Identical to the findings with ssDNA, MeP substitutions at the +2 position resulted in only small changes in the activation barrier (+/-0.3 kcal/mol), with little stereoselectivity ( approximately 4-fold). However, the H148A mutation destabilizes both the ground state and transition states by 2.4 and 4.3 kcal/mol, respectively. Thus, His148 is catalytic because it stabilizes the transition state to a greater extent (1.9 kcal/mol) than the ground state. Heteronuclear NMR studies established that His148 was neutral in the free enzyme at neutral pH, and in conformational exchange in a specific DNA complex containing uracil. We conclude that the +1 and +2 phosphate esters play identical catalytic roles in the reactions of single-stranded and double-stranded DNA substrates, and that His148 serves a catalytic role by positioning the substrate and catalytic water, or by an environmental effect.
Parker
Jared B
JB
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205-2185, USA.
Stivers
James T
JT
D000838:Anions / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005786:Gene Expression Regulation; D015394:Molecular Structure; D009154:Mutation; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D051981:Uracil-DNA Glycosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000838:Anions; D009943:Organophosphorus Compounds; D010710:Phosphates; C032627:methylphosphonic acid; D004247:DNA; D051981:Uracil-DNA Glycosidase