Mutations in the R-type pyruvate kinase gene and altered enzyme kinetic properties in patients with hemolytic anemia due to pyruvate kinase deficiency.

The biochemical properties of erythrocyte pyruvate kinase (PK) together with mutations found in the coding sequence of the R-PK gene in five patients with severe hemolytic anemia due to PK deficiency are described. The enzyme variants were designated PK 'Mosul' (homozygote), PK 'Bukarest', PK 'Hamburg', PK 'Köln', and PK 'Essen' (compound heterozygote). PK 'Mosul' showed normal positive cooperative substrate binding, PK 'Bukarest' exhibited non-cooperative behavior, and PK 'Hamburg' and PK 'Köln' displayed mixed cooperativity, whereas PK 'Essen' was negative cooperative. PK 'Mosul' was found to be homozygous for the mutation 1151 ACG to ATG, resulting in an amino acid substitution 384 Thr to Met. In one allele of PK 'Bukarest' a single nucleotide substitution GAG-TAG was found at nucleotide 721, causing a change of 241 Glu to a chain termination codon (PK 'Bukarest'). Additionally, in the second allele of this patient a point mutation at position 1594 (CGG-TGG) occurs, changing 532 Arg to Trp (PK 'Bukarest'). Direct sequencing showed the heterozygosity of the patient's mother (PK 'Bukarest'/normal) at position 721 and of the patient's father (PK 'Bukarest'/normal) at position 1594. A point mutation at position 1529 (CGA-CAA), causing an amino acid substitution 510 Arg-Gln, was identified in PK 'Hamburg' and PK 'Köln'. The second mutation in these variants was not detected. In PK 'Essen' no mutation in the coding sequence was found at all. Screening for the mutation at position 1529 in further compound heterozygote patients and in normal subjects of Western European origin showed that this exchange is a common mutation responsible for PK deficiency in this population.