Kinetic properties of mutant deoxyguanosine kinase in a case of reversible hepatic mtDNA depletion
Résumé
Deoxyguanosine kinase (DGUOK) is one of the two mitochondrial (mt) deoxynucleoside salvage pathway enzymes involved in precursor synthesis for mtDNA replication. DGUOK is responsible for the initial rate-limiting phosphorylation of the purine deoxynucleosides, using a nucleoside triphosphate as phosphate donor. Mutations in the DGUOK gene are associated with the hepato-specific and hepatocerebral forms of mtDNA depletion syndrome (MDS). We identified two missense mutations (N46S and L266R) in the DGUOK gene of a previously reported child, now 10-years-old, who presented with an unusual revertant phenotype of liver MDS. The kinetic properties of normal and mutant DGUOK were studied in mitochondrial preparations from cultured skin fibroblasts, using an optimized methodology. The N46S/L266R DGUOK showed 14% and 10% residual activity as compared with controls with deoxyguanosine and deoxyadenosine as phosphate acceptors, respectively. Similar apparent negative cooperativity in the binding of the phosphate acceptors to the wild-type enzyme was found for the mutant. In contrast, abnormal bimodal kinetics were shown with ATP as the phosphate donor, suggesting an impairment of the ATP binding mode at the phosphate donor site. No kinetic behaviors were found for two other patients with splicing defects or premature stop codon. This study represents the first characterization of the enzymatic kinetic properties of normal and mutant DGUOK in organello and our optimized protocol allowed us to demonstrate a residual activity in skin fibroblast mitochondria from a patient with a revertant phenotype of MDS. The residual DGUOK activity may play a crucial role in the phenotype reversal.
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