4ip7
Structure of the S12D variant of human liver pyruvate kinase in complex with citrate and FBP.
Structural highlights
DiseaseKPYR_HUMAN Defects in PKLR are the cause of pyruvate kinase hyperactivity (PKHYP) [MIM:102900; also known as high red cell ATP syndrome. This autosomal dominant phenotype is characterized by increase of red blood cell ATP.[1] Defects in PKLR are the cause of pyruvate kinase deficiency of red cells (PKRD) [MIM:266200. A frequent cause of hereditary non-spherocytic hemolytic anemia. Clinically, pyruvate kinase-deficient patients suffer from a highly variable degree of chronic hemolysis, ranging from severe neonatal jaundice and fatal anemia at birth, severe transfusion-dependent chronic hemolysis, moderate hemolysis with exacerbation during infection, to a fully compensated hemolysis without apparent anemia. FunctionKPYR_HUMAN Plays a key role in glycolysis (By similarity). Publication Abstract from PubMedDuring our efforts to characterize the regulatory properties of human liver pyruvate kinase (L-PYK), we have noted that the affinity of the protein for phosphoenolpyruvate (PEP) becomes reduced several days after cell lysis. A 1.8 A crystallographic structure of L-PYK with the S12D mimic of phosphorylation indicates that Cys436 is oxidized, the first potential insight into explaining the effect of "aging". Interestingly, the oxidation is only to sulfenic acid despite the crystal growth time period of 2 weeks. Mutagenesis confirms that the side chain of residue 436 is energetically coupled to PEP binding. Mass spectrometry confirms that the oxidation is present in solution and is not an artifact caused by X-ray exposure. Exposure of the L-PYK mutations to H(2)O(2) also confirms that PEP affinity is sensitive to the nature of the side chain at position 436. A 1.95 A structure of the C436M mutant of L-PYK, the only mutation at position 436 that has been shown to strengthen PEP affinity, revealed that the methionine substitution results in the ordering of several N-terminal residues that have not been ordered in previous structures. This result allowed speculation that oxidation of Cys436 and phosphorylation of the N-terminus at Ser12 may function through a similar mechanism, namely the interruption of an activating interaction between the nonphosphorylated N-terminus with the nonoxidized main body of the protein. Mutant cycles were used to provide evidence that mutations of Cys436 are energetically synergistic with N-terminal modifications, a result that is consistent with phosphorylation of the N-terminus and oxidation of Cys436 functioning through mechanisms with common features. Alanine-scanning mutagenesis was used to confirm that the newly ordered N-terminal residues were important to the regulation of enzyme function by the N-terminus of the enzyme (i.e., not an artifact caused by the introduced methionine substitution) and to further define which residues in the N-terminus are energetically coupled to PEP affinity. Collectively, these studies indicate energetic coupling (and potentially mechanistic similarities) between the oxidation of Cys436 and phosphorylation of Ser12 in the N-terminus of L-PYK. Energetic Coupling between an Oxidizable Cysteine and the Phosphorylatable N-Terminus of Human Liver Pyruvate Kinase.,Holyoak T, Zhang B, Deng J, Tang Q, Prasannan CB, Fenton AW Biochemistry. 2013 Jan 22;52(3):466-76. doi: 10.1021/bi301341r. Epub 2013 Jan 11. PMID:23270483[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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