Figure 4.

Phosphoenolpyruvate Accumulates in Cells with Low PYK Activity and Inhibits Triosephosphate Isomerase

(A) HILIC-MRM quantification of PEP (left panel) Standard addition of PEP to whole-cell methanol/water yeast extracts; and quantification of PEP separated by HILIC using multiple reaction monitoring (MRM). Quantification is demonstrated by linear regression, R² vales of >0.97 were obtained. (Right panel) HILIC-MRM quantification of PEP in TEF_pr-PYK1, CYC_pr-PYK1, and CYC_pr-PYK2 yeast; PEP is strongly accumulated in yeast with low PYK activity. Error bars, ±SD, n = 3.

(B) PEP inactivates yeast and mammalian TPI. Michaelis-Menten kinetics were determined for yeast TPI (left panel), human TPI (middle panel), and rabbit muscle TPI (right panel). To determine v_max and K_m, the TPI substrate gly3p was added in incremental doses (left y and lower x axis, blue; y values are normalized to total cellular protein [left and middle panel] or to the purified protein [right panel]). For all TPI isozymes, v_max is highlighted with a green dot in the saturation curve; K_m values are given in μM (right y and upper x axis, black). Inhibition of TPI activity demonstrated by addition of PEP in incremental concentrations; IC₅₀ values and the inhibitory constant K_i are given in μM, the Kcat in Mol^∗s⁻¹.

(C) Inactivation of pathogenic TPI alleles by PEP. TPI activity was assayed in transgenic yeast expressing human TPI (WT) or indicated pathogenic TPI alleles without or in the presence of 900 μmol PEP. Error bars, ±SD. Values within bars indicate the absolute enzyme activity in μmol/(min^∗μg protein).

(D) PYK activity does not change oxidant resistance in yeast expressing TPI_Ile170Val. The wild-type control, transgenic yeast expressing either PYK1 or PYK2, and either human TPI or human TPI_Ile170Val were spotted as serial dilutions onto SC media without or with diamide (left panel). Spot growth on different concentrations was analyzed with CellProfiler and normalized to the strain with highest PYK activity (right panel). Low PYK activity increased diamide tolerance if expressed in combination with human TPI, but there were no differences in TPI_Ile170Val-expressing yeast.