Transient State Kinetic Studies of the MutT-Catalyzed Nucleoside Triphosphate Pyrophosphohydrolase Reaction

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• 作者：Zuyong Xia ; Hugo F. Azurmendi ; and Albert S. Mildvan
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：November 22, 2005
• 年：2005
• 卷：44
• 期：46
• 页码：15334 - 15344
• 全文大小：179K
• 年卷期：v.44,no.46(November 22, 2005)
• ISSN：1520-4995

文摘

The MutT pyrophosphohydrolase, in the presence of Mg²⁺, catalyzes the hydrolysis ofnucleoside triphosphates by nucleophilic substitution at Ps/gifchars/beta2.gif" BORDER=0 ALIGN="middle">, to yield the nucleotide and PP_i. The bestsubstrate for MutT is the mutagenic 8-oxo-dGTP, on the basis of its K_m being 540-fold lower than thatof dGTP. Product inhibition studies have led to a proposed uni-bi-iso kinetic mechanism, in which PP_idissociates first from the enzyme-product complex (k₃), followed by NMP (k₄), leaving a product-bindingform of the enzyme (F) which converts to the substrate-binding form (E) in a partially rate-limiting step(k₅) [Saraswat, V., et al. (2002) Biochemistry 41, 15566-15577]. Single- and multiple-turnover kineticstudies of the hydrolysis of dGTP and 8-oxo-dGTP and global fitting of the data to this mechanism haveyielded all of the nine rate constants. Consistent with an "iso" mechanism, single-turnover studies withdGTP and 8-oxo-dGTP hydrolysis showed slow apparent second-order rate constants for substrate bindingsimilar to their k_cat/K_m values, but well below the diffusion limit (~10⁹ M^-1 s^-1): k_on^app = 7.2 × 10⁴ M^-1s^-1 for dGTP and k_on^app = 2.8 × 10⁷ M^-1 s^-1 for 8-oxo-dGTP. These low k_on^app values are fitted byassuming a slow iso step (k₅ = 12.1 s^-1) followed by fast rate constants for substrate binding: k₁ = 1.9× 10⁶ M^-1 s^-1 for dGTP and k₁ = 0.75 × 10⁹ M^-1 s^-1 for 8-oxo-dGTP (the latter near the diffusionlimit). With dGTP as the substrate, replacing Mg²⁺ with Mn²⁺ does not change k₁, consistent with theformation of a second-sphere MutT-M²⁺-(H₂O)-dGTP complex, but slows the iso step (k₅) 5.8-fold,and its reverse (k_-5) 25-fold, suggesting that the iso step involves a change in metal coordination, likelythe dissociation of Glu-53 from the enzyme-bound metal so that it can function as the general base.Multiple-turnover studies with dGTP and 8-oxo-dGTP show bursts of product formation, indicating partiallyrate-limiting steps following the chemical step (k₂). With dGTP, the slow steps are the chemical step (k₂= 10.7 s^-1) and the iso step (k₅ = 12.1 s^-1). With 8-oxo-dGTP, the slow steps are the release of the8-oxo-dGMP product (k₄ = 3.9 s^-1) and the iso step (k₅ = 12.1 s^-1), while the chemical step is fast (k₂= 32.3 s^-1). The transient kinetic studies are generally consistent with the steady state k_cat and K_m values.Comparison of rate constants and free energy diagrams indicate that 8-oxo-dGTP, at low concentrations,is a better substrate than dGTP because it binds to MutT 395-fold faster, dissociates 46-fold slower, andhas a 3.0-fold faster chemical step. The true dissociation constants (K_D) of the substrates from the E-formof MutT, which can now be obtained from k_-1/k₁, are 3.5 nM for 8-oxo-dGTP and 62 s/entities/mgr.gif">M for dGTP,indicating that 8-oxo-dGTP binds 1.8 × 10⁴-fold tighter than dGTP, corresponding to a 5.8 kcal/mollower free energy of binding.