MOLECULAR-BASES OF HEXOKINASE DEFICIENCY

Hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1; HK) defici ency is a rare disease where the predominant clinical effect is nonsph erocytic hemolytic anemia. We have previously shown that the only pati ent for which hexokinase deficiency has been so far investigated at mo lecular level is a double heterozygote carrying T-1667 --> C substitut ion on one HK type I allele and a 96 bp deletion (concerning nucleotid es 577 to 672 in the HK cDNA sequence) in the other allele. To investi gate 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 hexokinas e type I and two different mutants carrying the T --> C nucleotide sub stitution 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 h omogeneity, exhibited the same kinetic properties of human placenta he xokinase type I. Both mutant hexokinases were also expressed as solubl e recombinant proteins under the same conditions, but they showed an i mpaired catalytic activity with respect to the wild-type enzyme. In pa rticular, the T-1667 --> C substitution, causing the amino acid change from Leu(529) to Ser, is responsible for the complete loss of the hex okinase catalytic activity, while the 96 bp deletion causes a drastic reduction of the hexokinase activity. Taken together, both mutations e xplain the hexokinase deficiency found in the patient with the 'HK-Mel zo' variant. (C) 1997 Elsevier Science B.V.