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

Abstract

Escherichia coli GDP-mannose mannosyl hydrolase (GDPMH), a homodimer, catalyzes the hydrolysis of GDP-alpha-D-sugars to yield the beta-D-sugar and GDP by nucleophilic substitution with inversion at the C1' carbon of the 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 kcat values of 0.15 and 0.13 s(-1), respectively, at 22 degrees C and pH 7.5. Kinetic analysis of the Mn2+-activated enzyme yielded a K(m) of free Mn2+ of 3.9 +/- 1.3 mM when extrapolated to zero substrate concentration (K(a)Mn2+), which tightened to 0.32 +/- 0.18 mM when extrapolated 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) showed an order of magnitude decrease at saturating Mn2+. Such mutual tightening of metal and substrate binding suggests the formation of an enzyme-metal-substrate bridge complex. Direct Mn2+ binding studies, monitoring the concentration of free Mn2+ by EPR and of bound Mn2+ by its enhanced paramagnetic effect on the longitudinal relaxation rate of water 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 (epsilon(b)) of 11.5 +/- 1.2 indicates solvent access to the enzyme-bound Mn2+ ions. No cross relaxation was detected among the three bound Mn2+ ions, suggesting them to be separated by at least 10 A. Such studies also yielded a weak dissociation constant for the binary Mn2+-GDP-mannose complex (K1 = 6.5 +/- 1.0 mM) which significantly exceeded the kinetically determined K(m) values of Mn2+, indicating the true substrate to be GDP-mannose rather than its Mn2+ complex. Substrate binding 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 microM), was studied. Binding studies revealed a weak, binary E-GDP complex (K(D)(GDP) = 9.4 +/- 3.2 mM) which tightened approximately 500-fold in the presence of Mn2+ to yield a ternary E-Mn2+-GDP complex with a dissociation constant, K3(GDP) = 18 +/- 9 microM, which overlaps with the K(I)(GDP). The tight binding of Mn2+ to 0.7 +/- 0.2 site per enzyme subunit in the ternary E-Mn2+-GDP complex (K(A)' = 15 microM) and the tight binding of GDP to 0.8 +/- 0.1 site per enzyme subunit in the ternary E-Mg2+-GDP complex (K3 < 0.5 mM) indicate a stoichiometry close to 1:1:1 at the active site. The decrease in the enhancement factor of the ternary E-Mn2+-GDP complex (epsilon(T) = 4.9 +/- 0.4) indicates decreased solvent access to...

Functional characterization of the mammalian mRNA decapping enzyme hDcp2

“…Exceptions include the Escherichia coli GDP-mannose mannosyl hydrolase, which is equally efficient with both Mg 2+ and Mn 2+ (Legler et al 2002) (Dobrzanska et al 2002). hDcp2 is the second example of a Nudix protein whose activity is greatly enhanced with Mn 2+ as the divalent cation.…”

Quantitative characterization of the Mn2+ complexes of ADP and ATPγS by W-band ENDOR

“…A family of related enzymes are those involving the nucleotides GTP (guanosine triphosphate) and GDP (guanosine diphosphate). Examples are the protein Ras, which plays a central role asa molecular switch in cellular signal transduction [7], and the + Mn2~ GDP-mannose mannosyl hydrolase (GDPMH), which catalyzes the hydrolysis of GDP-cc-D-sugars to yield the 13-D-sugar and GDP [8]. Divalent metal binding to nucleotides is also highly relevant in RNA catalysis, where again Mn 2+ has been used to illuminate the role of the metal ions [9,10].…”

Mn2+-adenosine nucleotide complexes in the presence of the nitrogenase iron-protein: detection of conformational rearrangements directly at the nucleotide binding site by EPR and 2D-ESEEM (two-dimensional electron spin-echo envelope modulation spectroscopy)

“…In the present work we use cw-EPR (continuous-wave EPR) and pulsed EPR techniques to investigate the local metal-centre co-ordination environment of the Fe-protein nucleotide-binding site using the active nucleotide metal analogue, with paramagnetic Mn 2+ bound to the nucleotide in place of diamagnetic Mg 2+ . It also allows the application of ESEEM (electron spin-echo envelope modulation spectroscopy) techniques for the spectroscopic investigation of the local nucleotide-binding-site environment; this has previously been used successfully for the study of some other ATPases and GTPases [17][18][19][20][21][22][23][24]. ESEEM spectroscopy is a pulsed EPR method that can be used to study shf (superhyperfine) interactions that arise from the (usually) weak couplings of an electron-spin to nuclear-spin centres in its vicinity.…”