The DNA repair enzyme uracil DNA glycosylase (UDG) is a powerful N-glycohydrolase that cleaves the glycosidic bond of deoxyuridine in DNA. We have investigated the role of substrate binding energy in catalysis by systematically dismantling the optimal substrate Ap(+1)UpA(-1)pA(-2) by replacing the nucleotides at the +1, -1, or -2 position with a tetrahydrofuran abasic site nucleotide (D), a 3-hydroxypropyl phosphodiester spacer (S), a phosphate monoester (p), or a hydroxyl group (h). Contrary to previous reports; the minimal substrate for UDG is 2 ' -deoxyuridine (hUh). UDG has a significant catalytic efficiency (CE) for hUh of 4 x 10(7) M-1 [CE (k(cat)/K-m)(1/k(non)), where k(non) is the rate of the spontaneous hydrolysis reaction of hUh at 25 degreesC]. Addition of +1 and -1 phosphate monoanions to form pUp increases k(cat)/K-m, by 45-fold compared to that of hUh. The k(cat)/K-m for pUp, but not pU or Up, is found to decrease by 20-fold over the pH range of 6-9 with a pK(a) of 7.1, which is identical to the pK(a) values for deprotonation of the +1 and -1 phosphate groups determined by the pH dependence of the P-31 NMR chemical shifts. This pH dependence indicates that binding of the pUp tetraanion is disfavored, possibly due to unfavorable desolvation or electrostatic properties of the highly charged +1 and -1 phosphate groups. Addition of flexible hydroxypropyl groups to the +1 and -1 positions to make SpUpS increases k(cat)/K-m by more than 10(5)-fold compared to that of hUh, which is a 20-fold greater effect than observed with rigid D substituents in these positions (i.e., DpUpD). The -2 phosphoester or nucleotide is found to increase the reactivity of trimer substrates with rigid furanose rings or nucleotides in the fl and -1 positions by 1300--270000-fold (i.e., DpUpD --> DpUpDpA or ApUpA --> ApklpApA). In contrast, the -2 nucleotide provides only an g-fold rate enhancement when appended to the substrate containing the more flexible fl and -1 S substituents (SpUpS --> SpUpSpA). These context-dependent effects of a -2 nucleotide are interpreted in terms of a mechanism in which the binding energy of this "handle" is used drive the rigid +1 and -1 A or D substituents into their binding packets, resulting in a net catalytic benefit of -4.3 to -7.5 kcal/mol; Taken together, these results systematically track how UDG uses distant site binding interactions to produce an overall four billion-fold increase in CE compared to that of the minimal substrate hUh.
