Kinetic and Magnetic Resonance Studies of the Role of Metal Ions in the Mechanism of Escherichia coli GDP-mannose Mannosyl Hydrolase, an Unusual Nudix Enzyme

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• 作者：Patricia M. Legler ; H. Caroline Lee ; Jack Peisach ; and Albert S. Mildvan
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：April 9, 2002
• 年：2002
• 卷：41
• 期：14
• 页码：4655 - 4668
• 全文大小：171K
• 年卷期：v.41,no.14(April 9, 2002)
• ISSN：1520-4995

文摘

Escherichia coli GDP-mannose mannosyl hydrolase (GDPMH), a homodimer, catalyzes the hydrolysisof GDP-mages/gifchars/alpha.gif" BORDER=0>-D-sugars to yield the mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-D-sugar and GDP by nucleophilic substitution with inversion at the C1' carbon ofthe sugar [Legler, P. M., Massiah, M. A., Bessman, M. J., and Mildvan, A. S. (2000) Biochemistry 39, 8603-8608].GDPMH requires a divalent cation for activity such as Mn²⁺ or Mg²⁺, which yield similar k_cat values of 0.15 and 0.13s^-1, respectively, at 22 mages/entities/deg.gif">C and pH 7.5. Kinetic analysis of the Mn²⁺-activated enzyme yielded a K_m of free Mn²⁺ of3.9 ± 1.3 mM when extrapolated to zero substrate concentration (K_a^Mn2+), which tightened to 0.32 ± 0.18 mM whenextrapolated to infinite substrate concentration (K_m^Mn2+). Similarly, the K_m of the substrate extrapolated to zero Mn²⁺concentration (K_S^GDPmann = 1.9 ± 0.5 mM) and to infinite Mn²⁺ concentration (K_m^GDPmann = 0.16 ± 0.09 mM) showedan order of magnitude decrease at saturating Mn²⁺. Such mutual tightening of metal and substrate binding suggeststhe formation of an enzyme-metal-substrate bridge complex. Direct Mn²⁺ binding studies, monitoring the concentrationof free Mn²⁺ by EPR and of bound Mn²⁺ by its enhanced paramagnetic effect on the longitudinal relaxation rate ofwater protons (PRR), detected three Mn²⁺ binding sites per enzyme monomer with an average dissociation constant(K_D) of 3.2 ± 1.0 mM, in agreement with the kinetically determined K_a^Mn2+. The enhancement factor (mages/gifchars/epsilon.gif" BORDER=0 >_b) of 11.5 ±1.2 indicates solvent access to the enzyme-bound Mn²⁺ ions. No cross relaxation was detected among the three boundMn²⁺ ions, suggesting them to be separated by at least 10 Å. Such studies also yielded a weak dissociation constantfor the binary Mn²⁺-GDP-mannose complex (K₁ = 6.5 ± 1.0 mM) which significantly exceeded the kineticallydetermined K_m values of Mn²⁺, indicating the true substrate to be GDP-mannose rather than its Mn²⁺ complex. Substratebinding monitored by changes in ¹H-¹⁵N HSQC spectra yielded a dissociation constant for the binary E-GDP-mannose complex (K_S^GDPmann) of 4.0 ± 0.5 mM, comparable to the kinetically determined K_S value (1.9 ± 0.5 mM).To clarify the metal stoichiometry at the active site, product inhibition by GDP, a potent competitive inhibitor (K_I =46 ± 27 mages/entities/mgr.gif">M), was studied. Binding studies revealed a weak, binary E-GDP complex (K_D^GDP = 9.4 ± 3.2 mM)which tightened ~500-fold in the presence of Mn²⁺ to yield a ternary E-Mn²⁺-GDP complex with a dissociationconstant, K₃^GDP = 18 ± 9 mages/entities/mgr.gif">M, which overlaps with the K_I^GDP. The tight binding of Mn²⁺ to 0.7 ± 0.2 site per enzymesubunit in the ternary E-Mn²⁺-GDP complex (K_A' = 15 mages/entities/mgr.gif">M) and the tight binding of GDP to 0.8 ± 0.1 site perenzyme subunit in the ternary E-Mg²⁺-GDP complex (K₃ < 0.5 mM) indicate a stoichiometry close to 1:1:1 at theactive site. The decrease in the enhancement factor of the ternary E-Mn²⁺-GDP complex (mages/gifchars/epsilon.gif" BORDER=0 >_T = 4.9 ± 0.4) indicatesdecreased solvent access to the active site Mn²⁺, consistent with an E-Mn²⁺-GDP bridge complex. Fermi contactsplitting (4.3 ± 0.2 MHz) of the phosphorus signal in the ESEEM spectrum established the formation of an innersphere E-Mn²⁺-GDP complex. The number of water molecules coordinated to Mn²⁺ in this ternary complex wasdetermined by ESEEM studies in D₂O to be two fewer than on the average Mn²⁺ in the binary E-Mn²⁺ complexes,consistent with bidentate coordination of enzyme-bound Mn²⁺ by GDP. Kinetic, metal binding, and GDP bindingstudies with Mg²⁺ yielded dissociation constants similar to those found with Mn²⁺. Hence, GDPMH requires onedivalent cation per active site to promote catalysis by facilitating the departure of the GDP leaving group, unlike itshomologues the MutT pyrophosphohydrolase, which requires two, or Ap₄A pyrophosphatase, which requires three.