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 21 for activity and binds three to four ions tightly in the absence of ligands: K D 5 0.8 lM for one ion with a K D of 38 lM for the other Mg 21 ions. However, the enzyme requires 5-10 mM Mg 21 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 21 ; D26A, E39A, and E41A showed no significant change in the Mg 21 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 21 in the active site in the absence of Asp38. While lithium ion inhibition of the archaeal IMPase is very poor (IC 50~2 50 mM), the D38A enzyme has a dramatically enhanced sensitivity to Li 1 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.

Cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of an inositol monophosphatase family protein (SAS2203) fromStaphylococcus aureusMSSA476

“…However, in accordance with recent structural studies, the Li +sensitivity of IMPases can be related to the length and the flexibility of the active-site mobile loop containing the residues binding the third Mg 2+ ion (Johnson et al, 2001;Stieglitz et al, 2002;Li et al, 2010). In IMPases, the participation of the third Mg 2+ ion in phosphoester hydrolysis is crucial as it can activate the water nucleophile in order to initiate the reaction (Li et al, 2010). Li + , which has almost the same ionic radius as Mg 2+ , can effectively replace the latter, but owing to the difference in their preferred coordination number (four versus six) and the geometries of their coordination complexes (tetrahedral versus octahedral) Li + cannot place the water nucleophile in the proper position and hence the phosphatase activity is severely impaired.…”

“…The degree of Li + inhibition as well as the substrate specificity of this group of enzymes varies greatly depending on the source organism and the molecular basis of this variation is still a matter of controversy. However, in accordance with recent structural studies, the Li +sensitivity of IMPases can be related to the length and the flexibility of the active-site mobile loop containing the residues binding the third Mg 2+ ion (Johnson et al, 2001;Stieglitz et al, 2002;Li et al, 2010). In IMPases, the participation of the third Mg 2+ ion in phosphoester hydrolysis is crucial as it can activate the water nucleophile in order to initiate the reaction (Li et al, 2010).…”

Structural elucidation of the binding site and mode of inhibition of Li+ and Mg2+ in inositol monophosphatase

“…In human IMPase, the Mg1 binding site is consisting of Glu70, Asp90 and the carbonyl oxygen of Ile92 , and the Mg2 binding site is consisting of Asp90, Asp93 and Asp220 residues . The Mg3 binding site comprises a single amino acid (Glu65) in archaeal IMPase, corresponding to Glu70 in humans . Mg1 and Mg3 activate the catalytic water nucleophile (W1) (Fig.…”

“…However, these results were biased because of the assumed mechanism for two metal ion‐mediated phosphate hydrolysis and a lack of direct evidence. Another approach that may explain the Li + ‐induced inhibition suggests that the Mg3 site is the target of Li + binding ; however, this hypothesis suffers from several shortcomings. First, the mechanism cannot explain the ‘burst phase’ release of inositol during Li + inhibition .…”

Systems Biology Understanding of the Effects of Lithium on Cancer

“…The plausibility of such a possibility is exemplified by a functional, structural, and mutational study on an archaeal inositol monophosphatase. 55 The archaeal enzyme has high homology (30% identical, 50% similar) to its human counterpart and functions in the same magnesium-dependent manner. In this study it was…”