THE CONTRIBUTION OF LYSINE-36 TO CATALYSIS BY HUMAN MYOINOSITOL MONOPHOSPHATASE

The role of lysine residues in the catalytic mechanism of myo-inositol monophosphatase (EC 3.1.3.25) was investigated. The enzyme was comple tely inactivated by amidination with ethyl acetimidate or reductive me thylation with formaldehyde and cyanoborohydride. Activity was retaine d when the active site was protected with Mg2+, Li+, and D,L-myo-inosi tol 1-phosphate. Using radiolabeling, peptide mapping, and sequence an alysis, Lys-36 was shown to be the protected residue, which is respons ible for inactivation. Replacing Lys-36 with glutamine produced a muta nt protein, K36Q, with similar affinities for the substrate and the ac tivator Mg2+, but a 50-fold lower turnover number as compared to the w ild-type enzyme. Crystallographic studies did not indicate any gross s tructural changes in the mutant as compared to the native form. Initia l velocity data were best described by a rapid equilibrium ordered mec hanism with two Mg2+ binding before and a third one binding after the substrate. Inhibition by calcium was unaffected by the mutation, but i nhibition by lithium was greatly reduced and became noncompetitive. Th e pH dependence of catalysis and the solvent isotope effect on k(cat) are altered in the mutant enzyme. D,L-myo-Inositol 1-phosphate, 4-nitr ophenyl phosphate, and D-glucose 6-phosphate are cleaved at different rates by the wild-type enzyme, but with similar efficiency by K36Q. Al l data taken together are consistent with the hypothesis that modifyin g or replacing the lysine residue in position 36 decreases its polariz ing effect on one of the catalytic metal ions and prevents the efficie nt deprotonation of the metal-bound water nucleophile.