Probing the role of metal ions in the mechanism of inositol monophosphatase by site-directed mutagenesis.
Since inhibition of myo-inositol monophosphatase (EC 3.1.3.25) by lithium ions and the resulting attenuation of phosphatidylinositol cycle activity may be the mechanism by which lithium exerts its therapeutic effect in the treatment of manic depression, it is of great interest to understand the mechanism of the enzyme and how lithium and other metals interact with it. Divalent magnesium is essential for enzyme activity, whereas Li+ and high concentrations of Mg2+ act as uncompetitive inhibitors with respect to substrate. From the recently solved crystal structure of the human enzyme, several amino acid residues in the active site were targeted for mutagenesis studies. Nine single-residue substituted mutants were characterized with regard to catalytic parameters, Mg2+ dependence, and Li+ inhibition. In addition, a terbium fluorescence assay was developed to determine the metal binding properties of the wild-type and mutant enzymes. Although none of these mutations affected Km for substrate substantially, the mutations Glu70-->Gln, Glu70-->Asp, Asp90-->Asn and Thr95-->Ala, in which residues within coordinating distance of the active site metal were modified, all resulted in large reductions in catalytic activity. The position of Glu70 in the crystal structure further suggests that this residue may be involved in activating water for nucleophilic attack on the substrate. The mutations Lys36-->Ile, Asp90-->Asn, Thr95-->Ala, Thr95-->Ser, His217-->Gln, and Cys218-->Ala all resulted in parallel reductions in both lithium and magnesium affinity, suggesting that Li+ and Mg2+ share a common binding site.
Pollack
S J
SJ
Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Harlow, England.
Knowles
M R
MR
Atack
J R
JR
Broughton
H B
HB
Ragan
C I
CI
Osborne
S
S
McAllister
G
G
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry; D007700:Kinetics; D008094:Lithium / Q000378:metabolism* / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013725:Terbium / Q000378:metabolism
D013725:Terbium; D004247:DNA; D008094:Lithium; D010744:Phosphoric Monoester Hydrolases; C021822:myo-inositol-1 (or 4)-monophosphatase; D008274:Magnesium