Erythrocyte Adenylate Kinase Deficiency: First In-Depth Biochemical Characterization of the Y164C Mutant Enzyme Causing Hemolytic Anemia.

Adenylate kinases (AK, EC 2.7.4.3) are monomeric enzymes that catalyze the reversible transfer of the γ-phosphate group from a phosphate donor (normally ATP) to a phosphate acceptor (normally AMP) releasing two molecules of nucleoside diphosphates (ADP). Therefore, the AK enzymes play a crucial role in the synthesis of nucleotides that are required for a variety of cellular metabolic processes. At present, six isoforms of AK with different tissue localization and substrate specificity have been characterized in man. AK1 is a cytosolic enzyme, mainly expressed in skeletal muscle, brain and erythrocytes. Adenylate kinase deficiency is a rare red blood cell enzymopathy causing hemolysis; the genetic disorder is transmitted as an autosomal recessive trait. To date 7 different mutations have been reported. To analyze the mutations at the protein level, we have undertaken the expression, purification and enzymological characterization of the human AK1 in the wild-type and Y164C mutant form. The cDNA encoding the AK1 was obtained from a blood sample of a healthy donor, with normal AK1 activity. Maximal expression (about 100–120 mg of AK1 per liter of E.coli culture) was obtained after 6 hours of induction with 0.5 mM IPTG at 37°C. The recombinant enzyme was purified to homogeneity following a simple procedure. The specific activity was greater than 2000 U/mg. AK1 exhibited a relatively high stability (T₅₀ after 10 min of incubation, 53°C). Moreover, Mg-ATP protected enzyme activity from heat inactivation (T₅₀ about 20 degrees higher). The catalytic efficiency values towards ATP and AMP were essentially identical and similar to that of the authentic enzyme. The Y164C mutation has been found in an Italian patient with congenital hemolytic anemia and undetectable erythrocyte AK1 activity. By site directed-mutagenesis we have produced the Y164C mutant protein and we have performed the first in-depth functional analysis of AK1 causing hemolytic anemia. The mutant enzyme essentially exhibited K_m values unchanged, but drastic reductions of the catalytic efficiency towards both substrates (2 orders of magnitude). The Y164C mutant displayed little alterations in the thermostability properties. However, Mg-ATP failed to protect the enzyme activity from heat inactivation. Our data strongly suggest that the absence of AK1 activity observed in the patient homozygous for the Y164C mutation is due to inability of the enzyme to undergo conformational changes essential for the catalytic cycle.