PHOSPHOGLYCERATE KINASE DEFICIENCY: FUNCTIONAL PROPERTIES OF THE L89P, K191DEL, C316R AND A354P PATHOLOGICAL VARIANTS

Background. Phosphoglycerate kinase 1, (PGK1) the isoenzyme that catalyzes the first ATP-generating step in the glycolytic pathway, is encoded by a gene located on the X-chromosome, and is ubiquitously expressed. It is a relatively small monomeric molecule characterized by two hinge-bent domains, with a highly conserved structure. The N-terminal domain binds 1,3-bisphosphoglycerate (1,3BPG) or 3-phosphoglycerate (3PG), whereas the C-terminal domain binds Mg-ADP or Mg-ATP. During the catalytic cycle, the enzyme undergoes large conformational rearrangements, proceeding from an open form to a closed form. Mutations of the PGK1 gene result in an enzyme deficiency that is characterized by mild to severe haemolytic anaemia, neurological dysfunctions and myopathy. Patients rarely exhibit all the three clinical features. To date, 20 different mutations with worldwide distribution have been described. Aims. To gain knowledge on molecular bases of the haemolytic anaemia and/or neurological and muscolar dysfunctions associated to PGK deficiency, an in depth characterization of the PGK variants has been undertaken. The present study describes the biochemical characterization of L89P (c.266T>C), K191del (delAAG c.571>573), C316R (c.946T>C) and A354P (c.1060G>C) variant enzymes. K191del mutation was found in a patient affected by haemolytic anaemia; L89P, C316R and A354P mutations were identified in patients with haemolytic anaemia and neurological dysfunctions. Methods. Site-directed mutagenesis was used to introduce mutations into the PGK cDNA. All variants were expressed as recombinant forms in a microbial system and purified to homogeneity after a single anion exchange chromatographic step. Results. All mutations turned out to have detrimental effects on the molecular properties of PGK enzyme. All variants displayed high heat instability, being L89P, C316R and A354P the most affected (T50 values approximately 10°C lower than that of the wild type enzyme; t1/2 at 37°C, 9, 12 and 30 min, respectively, vs. >2h of the wild type). Differently from the wild type enzyme, all variants were not protected from heat inactivation by ATP or 3PG. In addition, A354P variant showed a Km value vs. 3PG 15-fold higher than that of the wild type enzyme, accounting for a 18-fold lowered catalytic efficiency. K191del and C316R enzymes exhibited 6- and 4-fold reduced kcat values. Conclusions. On the whole, these data primarily indicate that all mutations affect residues crucial for protein stability. Most probably, the inability to maintain proper folding in vivo makes the enzymes good targets for degradation by the ubiquitin-proteasome pathway, thus reducing their intracellular concentration, mainly in red blood cells. Moreover, the altered kinetic properties displayed by K191del, C316R and A354P variants render these enzymes unable to fulfil properly the catalytic cycle, contributing to lower the ATP generation.