Proton Donor in Yeast Pyruvate Kinase:  Chemical and Kinetic Properties of the Active Site Thr 298 to Cys Mutant

Abstract

The active site T298 residue of yeast pyruvate kinase (YPK), located in a position to serve potentially as the proton donor, was mutated to cysteine. T298C YPK was isolated and purified, and its enzymatic properties were characterized. Fluorescence and CD spectra indicate minor structural perturbations. A kinetic analysis of the Mg(2+)-activated enzyme demonstrates no catalytic activity in the absence of the heterotropic activator fructose 1,6-bisphosphate (FBP). In the presence of Mg(2+) and FBP, T298C has approximately 20% of the activity of wild-type (wt) YPK. The activator constant for FBP increases by 1 order of magnitude compared to this constant with the wt enzyme. T298C shows positive cooperativity by FBP with a Hill coefficient of 2.6 (wt, n(H,FBP) = 1). Mn(2+)-activated T298C behaves like Mn(2+)-activated wt YPK with a V(max) that is 20% of that for the wt enzyme with or without FBP. A pH-rate profile of T298C relative to that for wt YPK shows that pK(a,2) has shifted from 6.4 in wt to 5.5, indicating that the thiol group elicits an acidic pK shift. Inactivation of both wt and T298C by iodoacetate elicits a pseudo-first-order loss of activity with T298C being inactivated from 8 to 100 times faster than wt YPK. A pH dependence of the inactivation rate constant for T298C gives a value of 8.2, consistent with the pK for a thiol. Changes in fluorescence indicate that the T298C-Mg(2+) complex binds PEP, ADP, and both ligands together. This demonstrates that the lack of activity is not due to the loss of substrate binding but to the lack of ability to induce the proper conformational change. The mutation also induces changes in binding of FBP to all the relevant complexes. Binding of the metal and binding of PEP to the enzyme complexes are also differentially altered. Solvent isotope effects are observed for both wt and T298C. Proton inventory studies indicate that k(cat) is affected by a proton from water in the transition state and the effects are metal ion-dependent. The results are consistent with water being the active site proton donor. Active site residue T298 is not critical for activity but plays a role in the activation of the water and affects the pK that modulates catalytic activity.

Regulation of Yeast Pyruvate Kinase by Ultrasensitive Allostery Independent of Phosphorylation

“…However, prior studies indicated that FBP activates Cdc19 but not in a cooperative manner, i.e., with a Hill coefficient approximately equal to 1. These prior in vitro studies, however, measured enzymatic activity with FBP and PEP at non-physiological concentrations (Mesecar and Nowak, 1997a, b; Nowak and Bollenbach, 2001; Susan-Resiga and Nowak, 2004). While the cellular concentrations of PEP range from 0.03 to 0.8 mM, PEP was typically added in saturating concentrations of 20 mM to facilitate the enzyme activity measurements.…”

The contribution of two isozymes to the pyruvate kinase activity of Vibrio cholerae: One K+-dependent constitutively active and another K+-independent with essential allosteric activation

“…The software CHELATOR [22] was used to calculate ADP-Mg complexes and Mg 2+ free concentrations. In order to determine ADP-Mn complexes and Mn 2+ free concentrations, the K d of Mn 2+ was used [23]. The ionized PEP (PEP 3- ) concentrations were calculated considering a pK value of 6.3 [24].…”

Pyruvate kinase: Function, regulation and role in cancer

“…Tautomerization of enolpyruvate to the more stable keto form of pyruvate contributes to the favorable energetics of phosphate transfer from PEP to ADP. Tautomerization occurs when enolpyruvate accepts a proton from a water molecule that is held in position by conserved active site residues (T328 and S362 in humans) [27, 37, 38]. Following catalysis the products leave the active site, and neither substrate binding nor release of products is thought to be ordered [39].…”