Neubauer B, Lakomek M, Winkler H, Parke M, Hofferbert S, Schröter W
Universitäts-Kinderklinik, Göttingen, Germany.
Blood. 1991 May 1;77(9):1871-5.
The molecular alterations responsible for the characteristic enzyme abnormalities in pyruvate kinase (PK) deficiency were investigated in two unrelated children homozygous for PK deficiency. Both variant enzymes were characterized according to the recommendations of the International Committee for Standardization in Haematology. Genomic DNA was specifically amplified by the polymerase chain reaction. Normal and mutant alleles of the L-type PK gene were analyzed by nucleotide sequencing. Heterozygosity of the parents was confirmed by allele-specific oligonucleotide hybridization. In PK Linz a C to T base exchange at position 394 of the L-type PK gene was found. As a result, the 132nd amino acid of the mutant enzyme, arginine (CGC), is replaced by cysteine (TGC). The affected amino acid residue is located within the deduced active site of the protein and the enzyme variant shows strongly altered allosteric properties. PK Beirut shows a C for T substitution at position 1058, changing the 353 amino acid from threonine (ACG) to methionine (ATG). In contrast to PK Linz, this amino acid lies outside the deduced substrate binding site and kinetic parameters of PK Beirut are close to normal. Both enzyme variants show a markedly reduced specific activity and thermolability.
在两名丙酮酸激酶（PK）缺乏症纯合子的非亲缘儿童中，研究了导致丙酮酸激酶缺乏症特征性酶异常的分子改变。两种变异酶均按照国际血液学标准化委员会的建议进行了特征描述。通过聚合酶链反应对基因组DNA进行特异性扩增。通过核苷酸测序分析L型PK基因的正常和突变等位基因。通过等位基因特异性寡核苷酸杂交证实了父母的杂合性。在PK Linz中，发现L型PK基因第394位的C到T碱基交换。结果，突变酶的第132位氨基酸精氨酸（CGC）被半胱氨酸（TGC）取代。受影响的氨基酸残基位于推导的蛋白质活性位点内，并且该酶变体显示出变构性质的强烈改变。PK Beirut在第1058位显示C被T取代，将第353位氨基酸从苏氨酸（ACG）变为甲硫氨酸（ATG）。与PK Linz相反，该氨基酸位于推导的底物结合位点之外，并且PK Beirut的动力学参数接近正常。两种酶变体均显示出明显降低的比活性和热稳定性。
