Reconstructing the substrate for uracil DNA glycosylase: Tracking the transmission of binding energy in catalysis

Y. L. Jiang, J. T. Stivers

Research output: Contribution to journalArticlepeer-review

Abstract

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+1UpA-1pA-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 × 107 M-1 [CE = (kcat/Km)(1/knon), where knon is the rate of the spontaneous hydrolysis reaction of hUh at 25 °C]. Addition of +1 and -1 phosphate monoanions to form pUp increases kcat/Km by 45-fold compared to that of hUh. The kcat/Km for pUp, but not pU or Up, is found to decrease by 20-fold over the pH range of 6-9 with a pKa of 7.1, which is identical to the pKa values for deprotonation of the +1 and -1 phosphate groups determined by the pH dependence of the 31P 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 kcat/Km by more than 105-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 +1 and -1 positions by 1300-270000-fold (i.e., DpUpD → DpUpDpA or ApUpA → ApUpApA). In contrast, the -2 nucleotide provides only an 8-fold rate enhancement when appended to the substrate containing the more flexible +1 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 pockets, 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.

Original languageEnglish (US)
Pages (from-to)7710-7719
Number of pages10
JournalBiochemistry
Volume40
Issue number25
DOIs
StatePublished - Jun 26 2001

ASJC Scopus subject areas

  • Biochemistry

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