Wong Mun, Teng
(2008)
Identification of important residues for
catalysis and substrate specificity in
human choline kinase by site directed
mutagenesis.
Other.
Universiti Sains Malaysia.
(Submitted)
Abstract
Phosphatidylcholine (PtdCho) is a prominent constituent of eukaryotic and some
prokaryotic membranes. Choline kinase (CK), the initial enzyme of the COP-choline
pathway, mediates the conversion of choline to phosphorylcholine and is localized in
the supernatant fraction of cells. CK has been recognized as a new target for anticancer
therapy. To identify the amino acid residue of human CK (hCK) that is important for
catalysis, conserved aspartate at position 342 in hCKa2 was mutated to asparagine. The
mutant construct was successfully cloned into pET14b vector and overexpressed in
E. coli BL21 (DE3). The mutant protein D342NhCKa2 showed dramatic loss of activity,
only 12.46% of wild type protein activity remained. The Km for choline of the mutant
protein increased 1.18 folds while Km for ethanolamine increased 598 folds compared to
wild type. The Km and V max for ATP of mutant protein increased 3 and 4 folds
respectively. The increased Km suggested that the residue is important for the binding of
the substrates and play a role in catalysis. Mutation of aspartate 342 might also cause
the activity inhibition or disruption of homo-dimer complex in hCKa2.
Actions (login required)
 |
View Item |
