ERYTHROCYTE PYRUVATE-KINASE DEFICIENCY - THE INFLUENCE OF PHYSIOLOGICALLY IMPORTANT METABOLITES ON THE FUNCTION OF NORMAL AND DEFECTIVE ENZYMES

The dependence of the erythrocyte pyruvate kinase (PK)-catalyzed react ion on the glycolytic intermediates glucose-6-phosphate (Gluc-6-P), 2, 3-diphosphoglycerate (2,3-DPG) and the nucleotides ADP and ATP was stu died in normal individuals and 14 patients with PK deficiency. The Glu c-6-P concentrations in the erythrocytes are markedly elevated (4- to 6-fold) in 9 patients with severe hemolytic anemia compared to those 5 exhibiting a mild clinical course (up to 2-fold increased). 2,3-DPG i s elevated up to 2 times compared to the controls whereas the measured ADP and ATP only slightly deviate from the normal range. Control expe riments showed that these elevations of Gluc-6-P and 2,3-DPG do not de pend on the number of reticulocytes. In enzyme kinetic terms, Gluc-6-P shifts the Hill coefficent to smaller values, i.e. suppresses the pos itive cooperativity (sigmoidal reaction kinetics), found in normal and some of the mutant enzymes and shift the noncooperative enzymes of so me patients to an enzyme exhibiting negative cooperativity. The negati ve cooperativity already present in the enzymes of some of the patient s suffering from severe hemolytic anemia becomes more pronounced upon addition of Gluc-6-P. Apparently 2,3-DPG acts as an antagonist to Gluc -6-P in increasing the Hill coefficient, i.e. enhancing the positive c ooperativity of the normal enzyme. It shifts the hyperbolic patients' enzymes to a sigmoidal reaction type and the enzymes of those patients with negative cooperativity to a hyperbolic type. ADP and ATP show a similar behavior as 2,3-DPG, but additionally inhibit the enzyme at hi gher concentrations. The influence of all four phosphates on the Micha elis constant varies depending on the type of cooperativity, in some c ases increasing and in some cases decreasing K-0.5PEP. With 7 of the p atients, all of them with severe clinical course, a genetic analysis o f their R-type PK gene was performed and genetic defects have been ide ntified in the coding sequence. The found changes in the amino acid se quence and their corresponding location in the tertiary structure of t he PK subunit can satisfactorily explain the alterations of the regula tory properties of the mutant enzymes thus allowing to establish a goo d correlation between altered structural and functional properties of the deficient enzyme and the severeness of the course of the disease.