Molecular bases of hexokinase deficiency

Hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1; HK) deficiency is a rare disease where the predominant clinical effect is nonspherocytic hemolytic anemia. We have previously shown that the only patient for which hexokinase deficiency has been so far investigated at molecular level is a double heterozygote carrying a T1667 --> C substitution on one HK type I allele and a 96 bp deletion (concerning nucleotides 577 to 672 in the HK cDNA sequence) in the other allele. To investigate whether these mutations found in the patient with the hexokinase variant referred to as 'HK-Melzo' could be associated with hexokinase deficiency, we have expressed in E. coli the wild-type human hexokinase type I and two different mutants carrying the T --> C nucleotide substitution at position 1667 and the nt 577-672 deletion, respectively. Wild-type human recombinant hexokinase is expressed in bacterial cells as a soluble catalytically active enzyme that, upon purification to homogeneity, exhibited the same kinetic properties of human placenta hexokinase type I. Both mutant hexokinases were also expressed as soluble recombinant proteins under the same conditions, but they showed an impaired catalytic activity with respect to the wild-type enzyme. In particular, the T1667 --> C substitution, causing the amino acid change from Leu529 to Ser, is responsible for the complete loss of the hexokinase catalytic activity, while the 96 bp deletion causes a drastic reduction of the hexokinase activity. Taken together, both mutations explain the hexokinase deficiency found in the patient with the 'HK-Melzo' variant.