Mobile loop mutations in an archaeal inositol monophosphatase: modulating three-metal ion assisted catalysis and lithium inhibition

Abstract

The inositol monophosphatase (IMPase) enzyme from the hyperthermophilic archaeon Methanocaldococcus jannaschii requires Mg(2+) for activity and binds three to four ions tightly in the absence of ligands: K(D) = 0.8 muM for one ion with a K(D) of 38 muM for the other Mg(2+) ions. However, the enzyme requires 5-10 mM Mg(2+) for optimum catalysis, suggesting substrate alters the metal ion affinity. In crystal structures of this archaeal IMPase with products, one of the three metal ions is coordinated by only one protein contact, Asp38. The importance of this and three other acidic residues in a mobile loop that approaches the active site was probed with mutational studies. Only D38A exhibited an increased kinetic K(D) for Mg(2+); D26A, E39A, and E41A showed no significant change in the Mg(2+) requirement for optimal activity. D38A also showed an increased K(m), but little effect on k(cat). This behavior is consistent with this side chain coordinating the third metal ion in the substrate complex, but with sufficient flexibility in the loop such that other acidic residues could position the Mg(2+) in the active site in the absence of Asp38. While lithium ion inhibition of the archaeal IMPase is very poor (IC(50) approximately 250 mM), the D38A enzyme has a dramatically enhanced sensitivity to Li(+) with an IC(50) of 12 mM. These results constitute additional evidence for three metal ion assisted catalysis with substrate and product binding reducing affinity of the third necessary metal ion. They also suggest a specific mode of action for lithium inhibition in the IMPase superfamily.

Figure 1

Ribbon diagram of mobile loop and active site elements showing acidic residues mutated, the three activating metal ions (indicated by Met1, Met2, and Met3), and product inorganic phosphate. Note that Asp38 is a direct ligand of the third metal ion.

Figure 2

(A) Heat released (μcal/s) upon M. jannaschii IMPase binding aliquots of Mg²⁺; (B) Enthalpy (kcal/mol) of binding each aliquot of Mg²⁺ as a function of ratio of Mg²⁺ to IMPase monomer.

Figure 3

Far-UV CD spectrum for the M. jannaschii IMPase in the absence of Mg²⁺ (thick solid line) and with 10 μM (—) and 100 μM (^{_ _ _ _}) Mg²⁺ added. The inset shows the difference spectrum showing the increase in β-sheet after adding 10 μM Mg²⁺.

Figure 4

Kinetic parameters for recombinant M. jannaschii IMPase (WT) and mobile loop mutants: (A) K_D (mM) for cofactor Mg²⁺ evaluated by varying Mg²⁺ concentration at 2 mM D-I-1-P (a concentration chosen to be significantly above K_m for that substrate); (B) K_m (mM) for D-I-1-P substrate evaluated at fixed Mg²⁺ (10 mM for WT IMPase and 50 mM for Mg²⁺-impaired mutants); (C) k_cat (s⁻¹) extrapolated from V_max; (D) k_cat/K_m (M⁻¹s⁻¹). Bars indicate errors on each extracted kinetic parameter.

Figure 5

Monovalent cation inhibition of MJ0109 FBPase activity, (A) Effect of LiCl on MJ0109 D38A (filled circle) and wildtype (open square) activity towards 2 mM FBP with 10 mM MgCl₂; (B) Effect of LiCl (circles) and KCl (squares) on wildtype MJ0109 FBPase activity towards 0.5 mM FBP in the presence of 2 mM (filled symbols) or 10 mM (open symbols) MgCl₂.

Figure 6

(A) Electron density map for MJ0109 D38A mobile loop and active site shown in green with the model for the protein backbone as green ribbon. In blue is shown the canonical MJ0109 loop structure for wild type protein. Note the shift in the mobile loop away from the active site; (B) Model for D38A with Li⁺ bound at the active site in the place of the third Mg²⁺ ion. The loop is proposed to orient towards this ion via a water molecule rather than the direct contact seen in the wild type structure.