The contribution of lysine-36 to catalysis by human myo-inositol monophosphatase.
The role of lysine residues in the catalytic mechanism of myo-inositol monophosphatase (EC 3.1.3.25) was investigated. The enzyme was completely inactivated by amidination with ethyl acetimidate or reductive methylation with formaldehyde and cyanoborohydride. Activity was retained when the active site was protected with Mg2+, Li+, and D,L-myo-inositol 1-phosphate. Using radiolabeling, peptide mapping, and sequence analysis, Lys-36 was shown to be the protected residue, which is responsible for inactivation. Replacing Lys-36 with glutamine produced a mutant protein, K36Q, with similar affinities for the substrate and the activator Mg2+, but a 50-fold lower turnover number as compared to the wild-type enzyme. Crystallographic studies did not indicate any gross structural changes in the mutant as compared to the native form. Initial velocity data were best described by a rapid equilibrium ordered mechanism with two Mg2+ binding before and a third one binding after the substrate. Inhibition by calcium was unaffected by the mutation, but inhibition by lithium was greatly reduced and became noncompetitive. The pH dependence of catalysis and the solvent isotope effect on kcat are altered in the mutant enzyme. D,L-myo-Inositol 1-phosphate, 4-nitrophenyl phosphate, and D-glucose 6-phosphate are cleaved at different rates by the wild-type enzyme, but with similar efficiency by K36Q. All data taken together are consistent with the hypothesis that modifying or replacing the lysine residue in position 36 decreases its polarizing effect on one of the catalytic metal ions and prevents the efficient deprotonation of the metal-bound water nucleophile.
Ganzhorn
A J
AJ
Marion Merrel Research Institute, Strasbourg, France. AxelGanzhorn@mmd.com
Lepage
P
P
Pelton
P D
PD
Strasser
F
F
Vincendon
P
P
Rondeau
J M
JM
D001483:Base Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D017931:DNA Primers; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D017931:DNA Primers; D010744:Phosphoric Monoester Hydrolases; C021822:myo-inositol-1 (or 4)-monophosphatase; D008239:Lysine