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. 2013 Jan 22;52(3):466-76.
doi: 10.1021/bi301341r. Epub 2013 Jan 11.

Energetic coupling between an oxidizable cysteine and the phosphorylatable N-terminus of human liver pyruvate kinase

Todd Holyoak  1 , Bing Zhang, Junpeng Deng, Qingling Tang, Charulata B Prasannan, Aron W Fenton
Affiliations

Energetic coupling between an oxidizable cysteine and the phosphorylatable N-terminus of human liver pyruvate kinase

Todd Holyoak et al. Biochemistry. .

Abstract

During 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 Å 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 H₂O₂ also confirms that PEP affinity is sensitive to the nature of the side chain at position 436. A 1.95 Å 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.

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Figures

Figure 1
Figure 1
A time/aging dependent influence on the affinity of L-PYK for PEP. A) The initial velocity derived Kapp-PEP for wild type L-PYK as a function of storage time after purification (36 hrs. after cell lysis are required for purification). The line represents the data trend. B) The influence of “aging” on allosteric properties elicited by Fru-1,6-BP (squares) and by alanine (circles). The influence of ageing appears to primarily impact PEP affinity since the allosteric properties (the distance between the plateaus at low and high effector concentrations) is relatively unchanged for freshly purified (solid symbols) vs. “aged” (open symbols) enzyme. Lines represent the best fits to Equation 1.
Figure 2
Figure 2
A) Aberrant, unmodeled density adjacent to Cys436. 2Fo-Fc density rendered at 1.2σ and Fo-Fc density rendered at 2.5σ are illustrated as a blue and a green mesh, respectively. The dashed black line indicates a distance of 2.3Å from the center of the unmodeled density to the sulfur of C436. B) Spatial relationship of Cys436 (black spacefill) to other structural features. The four subunits of the hL-PYK homotetramer are in red, green, blue, and gray. The N-terminus of the red subunit is in cyan as determined by the C436M structure reported in this study. Citrate in the active site is in orange spacefill. Fru-1,6-BP is displayed in the top two subunits in magenta spacefill, but (although present in the structure) is not displayed in the two bottom subunits.
Figure 3
Figure 3
Sensitivity of Kapp-PEP for the wild type (black, filled circles) and C436A (red) L-PYK proteins to short (8 min.) incubation with H2O2. Exposure to concentrations up to 100 mM did not alter Vmax activity (data not shown). Wild type protein (black, open squares) in the presence of 2 mM DTT was included as a control. Data collection was replicated by multiple individuals using different protein preparations. Although general features were maintained in replicates (i.e. the wild type protein being much more sensitive to H2O2 as compared to C436A), there was considerable variability between protein preps in the Kapp-PEP determined for a given protein at a given H2O2 concentration. A representative comparison is included here.
Figure 4
Figure 4
N-terminal residues (yellow stick) that become ordered as a result of the C436M (red stick) mutation. Specifically, residues 18–24 (QELGTAF) are ordered as a result of the mutation. The side-chains of residues Leu20, Phe24, and Phe25 from the N-terminus interact with methionine at position 436 of the same subunit (blue). The newly ordered residues cross a subunit interface to interact with a second subunit (purple).
Figure 5
Figure 5
The impact of an N-terminus alanine scan on the ΔG associated with Ka-PEP. A) The impact of single residues substituted with alanine. Filled circles represent a comparison between measurements for mutant protein with that from wild type protein. Residues that are alanine or glycine in the wild type structure were not probed. Therefore, the wild type data used to represent Ka-PEP at these positions are represented by open circles to clearly distinguish data for mutant proteins. B) The impact of substituting two consecutive positions, both with alanine. C) The impact of substituting three consecutive positions with alanine. D) The overlay of the three data sets included in panels A, B, and C. In all panels, data are compared to that of the wild type protein. A positive deviation from zero on the y-axis indicates reduced PEP affinity; a negative value is improved PEP affinity. Panel D includes both the sum of effects caused by individual mutations (sum of Δ ΔG values for the two single alanine mutations; black) and the difference between the wild type value and that observed for the protein with both alanine substitutions of interest inserted simultaneously (Δ ΔG values for double alanine window mutations: red). As a means of simplifying panels, error bars are only included in panel A. However, error estimates are included for all data in the supplemental material. Green boxes are included in panels D and E to emphasize areas of interest.
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