Enzymes: galactokinase (EC 18.104.22.168).
Functional analysis of disease-causing mutations in human galactokinase
Article first published online: 24 MAR 2003
European Journal of Biochemistry
Volume 270, Issue 8, pages 1767–1774, April 2003
How to Cite
Timson, D. J. and Reece, R. J. (2003), Functional analysis of disease-causing mutations in human galactokinase. European Journal of Biochemistry, 270: 1767–1774. doi: 10.1046/j.1432-1033.2003.03538.x
- Issue published online: 25 MAR 2003
- Article first published online: 24 MAR 2003
- (Received 19 December 2002, revised 29 January 2003, accepted 24 February 2003)
- GHMP family kinase;
Galactokinase (EC 22.214.171.124) catalyzes the first committed step in the catabolism of galactose. The sugar is phosphorylated at position 1 at the expense of ATP. Lack of fully functional galactokinase is one cause of the inherited disease galactosemia, the main clinical manifestation of which is early onset cataracts. Human galactokinase (GALK1) was expressed in and purified from Escherichia coli. The recombinant enzyme was both soluble and active. Product inhibition studies showed that the most likely kinetic mechanism of the enzyme was an ordered ternary complex one in which ATP is the first substrate to bind. The lack of a solvent kinetic isotope effect suggests that proton transfer is unlikely to be involved in the rate determining step of catalysis. Ten mutations that are known to cause galactosemia were constructed and expressed in E. coli. Of these, five (P28T, V32M, G36R, T288M and A384P) were insoluble following induction and could not be studied further. Four of the remainder (H44Y, R68C, G346S and G349S) were all less active than the wild-type enzyme. One mutant (A198V) had kinetic properties that were essentially wild-type. These results are discussed both in terms of galactokinase structure-function relationships and how these functional changes may relate to the causes of galactosemia.