Energetic Coupling between an Oxidizable Cysteine and the Phosphorylatable N-Terminus of Human Liver Pyruvate Kinase

Abstract

During our efforts to characterize regulatory properties of human liver pyruvate kinase (L-PYK), we have noted that the affinity of the protein for PEP becomes reduced after several days post cell lysis. A 1.8Å 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 two-week time of crystal growth. Mutagenesis confirms that the 436 side-chain is energetically coupled to PEP binding. Mass spectrometry confirms that the oxidation is present in solution and is not an artifact due to X-ray exposure. Exposure to the L-PYK mutations to H2O2 also confirms that PEP affinity is sensitive to the nature of the side-chain at position 436. A 1.95Å structure of the C436M mutation of L-PYK, the only mutation at position 436 identified 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 a 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 non-phosphorylated N-terminus with the non-oxidized 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 due to 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.

Are all regions of folded proteins that undergo ligand‐dependent order–disorder transitions targets for allosteric peptide mimetics?

“…Within this binding motif Faustova et al found that many single amino acid substitutions could be accommodated with only moderate influences on PEP affinity; the primary exception was a mutation that altered the side-chain charge at position 9 (25). These findings are consistent with observation from studies in my laboratory that each residue in the 7–10 binding motif contributing to binding affinity, rather than the motif working as an all-or-none cooperative unit (28). Further into the N-terminus, Leu20, Phe24, and Phe25 all make favorable interactions with the side-chain of position 436 (28).…”

“…These findings are consistent with observation from studies in my laboratory that each residue in the 7–10 binding motif contributing to binding affinity, rather than the motif working as an all-or-none cooperative unit (28). Further into the N-terminus, Leu20, Phe24, and Phe25 all make favorable interactions with the side-chain of position 436 (28). Therefore, it seems likely that oxidation of Cys436 interferes with the ordering of additional N-terminal residues as compared to those that become disordered as a result of phosphorylation at Ser12.…”

“…Recent studies directed at understanding the molecular mechanism of this N-terminal regulation include a truncation series, designed point mutations surrounding the phosphorylation site, alanine-scanning mutagenesis using single, double consecutive, and triple consecutive alanines, mutant cycle analysis, and crystallographic studies of mutant protein (25–28). Data from these studies are consistent with the N-terminus binding to the main body of the protein in the absence of phosphorylation (or oxidation).…”

“…Phosphorylation at Ser12 and oxidation at Cys436 on the main body of the protein both prevent this activating interaction to result in the apparent modification-dependent reduction in PEP affinity (Figure 1). There is now sufficient information to assign detailed functions to residues in the 25 amino acid N-terminus of hL-PYK: $$\text{MEGPAG}\underset{¯}{YLRR}\text{A}\mathbf{S}\text{VAQLTQELGTAFF}\dots \dots$$ The initial six residues have no role in energetic coupling with PEP binding (26, 28). Residues 7–10 define a binding motif, although it is still unclear exactly where on the main body of the protein this motif docks.…”

Distinguishing the Interactions in the Fructose 1,6-Bisphosphate Binding Site of Human Liver Pyruvate Kinase That Contribute to Allostery

“…However, there are no differences in the actual electron densities for any of the co-crystallized structures. We recently solved a 1.85Å structure of hL-PYK with Fru-1,6-BP bound (14). The improved resolution confirms that Fru-1,6-BP binds to hL-PYK with the 1’-phosphate directed towards Arg501 of hL-PYK (Figure 1), the same binding orientation found in yeast-PYK, human M 2 -PYK, and human R-PYK.…”