Structural Enzymology of Li 1 -sensitive/Mg 21 - dependent Phosphatases S. Patel 1 , M. Martı ´nez-Ripoll 2 , Tom L. Blundell 1 and A. Albert 2 * 1 Department of Biochemistry University of Cambridge Tennis Court Road, Cambridge CB2 1GA, UK 2 Grupo de Cristalografı ´a Macromolecular y Biologı ´a Estructural, Instituto de Quı ´mica Fı ´sica “Rocasolano” Consejo Superior de Investigaciones Cientı ´ficas Serrano 119, E-28006 Madrid Spain Li þ -sensitive/Mg 2þ -dependent phosphatases have attracted considerable attention since they have been proposed as targets for lithium therapy in the treatment of manic-depressive patients. The members of this enzyme superfamily display low levels of sequence identity while possessing a common fold and active site. Extensive structural and biochemical data demonstrate the direct involvement of two metal ions in catalysis, and show that lithium exerts its inhibitory action by blocking the products at the active site. By exploiting the different inhibitory properties of magnesium and calcium, we have been able to solve the X-ray structures of the Li þ -sensitive/Mg 2þ -dependent 3 0 -phosphoadenosine-5 0 -phospha- tase in complex with its substrate and with its products. The structural comparison of these complexes provides a 3D picture of the different stages of the catalytic cycle. This gives new insights into the under- standing of the biological function of this group of enzymes and their lithium inhibition, and should assist in the design of improved inhibitors of therapeutic value. q 2002 Elsevier Science Ltd. All rights reserved Keywords: inositol; 3 0 -phosphoadenosine-5 0 -phosphate; lithium therapy; protein crystallography *Corresponding author Introduction Li þ -sensitive/Mg 2þ -dependent phosphatases (LiMgPPases) comprise a group of evolutionarily related enzymes that exhibit an absolute require- ment for divalent metal ions and are inhibited by lithium. 1 They are well characterized and they include putative targets for lithium therapy in the treatment of manic-depressive patients. 2 LiMgPPases include fructose-1,6-bisphosphate 1-phosphatases (FBPases), inositol polyphosphate 1-phosphatases (IPPases), inositol monophospha- tases (IMPases), 3 0 -phosphoadenosine-5 0 -phospha- tases (PAPases) and enzymes acting on both inositol-1,4-bisphosphate and PAP (PIPases). 3 Extensive structural and kinetic investigations 4,5 show that they possess a common structural core with the active site, including two or three metal-binding sites, lying between a þ b and a/b domains. The catalytic mechanism of LiMgPPases has been the subject of discussion for many years. Of particular interest is the question as to which of the bound metals provides the activation of the nucleophilic water molecule. Bone et al. suggested that the metal bound at site 1 would chelate the water molecule and that the mechanism would proceed via in-line nucleophilic displacement with inversion of configuration at phosphate. 6 Bone et al. suggested that a magnesium ion at site 1 and a Thr residue are responsible for the activation of the water molecule. 6 In this mechanism, another magnesium ion at site 2 is assumed to assist in phosphate coordination and charge stabilization during the transition state. On the other hand, Gani & Wilkie suggested that the activation of the nucleophilic water molecule occurs at site 2 and that the mechanism proceeds via adjacent associ- ation followed by pseudo-rotation with retention of configuration. 7 The determination of the stereo- chemical course of the reaction completely ruled out this mechanism. 8 However, Gani and 0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved Present address: S. Patel, Astex Technology Ltd, 250 Cambridge Science Park, Milton Road, Cambridge, CB4 0WE, UK. E-mail address of the corresponding author: xalbert@iqfr.csic.es Abbreviations used: LiMgPPase, Li þ -sensitive/Mg 2þ - dependent phosphatase; FBPase, fructose-1,6-bisphos- phate 1-phosphatase; IPPase, inositol polyphosphate 1-phosphatase; IMPase, inositol monophosphatase; PAP, 3 0 -phosphoadenosine-5 0 -phosphate; PAPase, 3 0 -phospho- adenosine-5 0 -phosphatases; PAPS, 3 0 -phosphoadenosine- 5 0 -phosphosulphate; PIPases, enzymes acting on both inositol-1,4-bisphosphate and PAP; CSD, the Cambridge Structural Database. doi:10.1016/S0022-2836(02)00564-8 available online at http://www.idealibrary.com on B w J. Mol. Biol. (2002) 320, 1087–1094