title: Structure and function of human erythrocyte pyruvate kinase. Molecular basis of nonspherocytic hemolytic anemia.
abstract: Deficiency of human erythrocyte isozyme (RPK) is, together with glucose-6-phosphate dehydrogenase deficiency, the most common cause of the nonspherocytic hemolytic anemia. To provide a molecular framework to the disease, we have solved the 2.7 A resolution crystal structure of human RPK in complex with fructose 1,6-bisphosphate, the allosteric activator, and phosphoglycolate, a substrate analogue, and we have functionally and structurally characterized eight mutants (G332S, G364D, T384M, D390N, R479H, R486W, R504L, and R532W) found in RPK-deficient patients. The mutations target distinct regions of RPK structure, including domain interfaces and catalytic and allosteric sites. The mutations affect to a different extent thermostability, catalytic efficiency, and regulatory properties. These studies are the first to correlate the clinical symptoms with the molecular properties of the mutant enzymes. Mutations greatly impairing thermostability and/or activity are associated with severe anemia. Some mutant proteins exhibit moderate changes in the kinetic parameters, which are sufficient to cause mild to severe anemia, underlining the crucial role of RPK for erythrocyte metabolism. Prediction of the effects of mutations is difficult because there is no relation between the nature and location of the replaced amino acid and the type of molecular perturbation. Characterization of mutant proteins may serve as a valuable tool to assist with diagnosis and genetic counseling.
authors.affiliation: Dipartimento di Genetica e Microbiologia, Università di Pavia, via Abbiategrasso 207, 27100 Pavia, Italy. giovale@unipv.it
authors.forename: Giovanna
authors.initials: G
authors.lastname: Valentini
authors.forename: Laurent R
authors.initials: LR
authors.lastname: Chiarelli
authors.forename: Riccardo
authors.initials: R
authors.lastname: Fortin
authors.forename: Manuela
authors.initials: M
authors.lastname: Dolzan
authors.forename: Alessandro
authors.initials: A
authors.lastname: Galizzi
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Abraham
authors.forename: Changqing
authors.initials: C
authors.lastname: Wang
authors.forename: Paola
authors.initials: P
authors.lastname: Bianchi
authors.forename: Alberto
authors.initials: A
authors.lastname: Zanella
authors.forename: Andrea
authors.initials: A
authors.lastname: Mattevi
mesh_terms: D000494:Allosteric Regulation; D000746:Anemia, Hemolytic, Congenital Nonspherocytic / Q000235:genetics; D020134:Catalytic Domain; D003461:Crystallography; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D016296:Mutagenesis; D011487:Protein Conformation; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000502:physiology; D013329:Structure-Activity Relationship
chemical_list: D011770:Pyruvate Kinase