The Proton Transfer Step Catalyzed by Yeast Pyruvate Kinase

Abstract

The nature of the proton donor to the C-3 of the enolate of pyruvate, the intermediate in the reaction catalyzed by yeast pyruvate kinase, was investigated by sitedirected mutagenesis and physical and kinetic analyses. Thr-298 is correctly located to function as the proton donor. T298S and T298A were constructed and purified. Both mutants are catalytically active with a decrease in k cat and k cat /K m,PEP . Mn 2؉ -activated T298S and T298A do not exhibit homotropic kinetic cooperativity with phosphoenolpyruvate (PEP) in the absence of fructose 1,6-bisphosphate, although PEP binding to enzyme-Mn 2؉ is cooperative. The pH dependence of k cat for T298A indicates the loss of pK a,2 ‫؍‬ 6.4 -6.9. Thr-298 affects the ionization (pK a Ϸ6.5) responsible for modulation of k cat . Fluorescence studies show altered dissociation constants of ligands to each enzyme complex upon Thr-298 mutations. The rates of the phosphoryl transfer and proton transfer steps in the pyruvate kinase-catalyzed reaction are altered; pyruvate enolization is affected to a greater extent. Proton inventory studies demonstrate solvent isotope effects on k cat and k cat /K m,PEP . Fractionation factors are metal-dependent and significantly <1. The data suggest that a water molecule in a water channel is the direct proton donor to enolpyruvate and that Thr-298 affects a late step in catalysis.Yeast pyruvate kinase (YPK) 1 (EC 2.7.1.4.0) is a key regulatory enzyme in glycolysis that catalyzes the phosphoryl transfer from phosphoenolpyruvate (PEP) to ADP to yield pyruvate and ATP. The reaction requires both monovalent and divalent cations, normally K ϩ and Mg 2ϩ or Mn 2ϩ . Fructose 1,6-bisphosphate (Fru-6-P 2 ) is the heterotropic activator of YPK. The net reaction catalyzed by YPK is the sum of at least two partial reactions. Phosphoryl transfer from PEP to M(II)ADP occurs by an apparent S n 2 mechanism with inversion of configuration at the phosphoryl group to yield the enolate of pyruvate and M(II)ATP (1). In the second partial reaction, a proton donor at the active site stereospecifically protonates the C-3 of enolpyruvate at the 2-si face of the double bond to form ketopyruvate (2-4). The enolate of pyruvate, the common species in both partial reactions, is a tightly bound intermediate in the net reaction catalyzed by PK (5). The two-step character of the net PK reaction is demonstrated by the ability of PK to catalyze the enolization of bound pyruvate without phosphoryl transfer (6 -8). Muscle PK requires a group such as inorganic phosphate, methyl phosphonate, or fluorophosphate as a cofactor (6, 7). YPK requires ATP as a cofactor for this activity (8). PK will also catalyze the ketonization of enolpyruvate, generated in situ subsequent to the hydrolysis of PEP catalyzed by alkaline phosphatase (3).Initial crystallographic data from cat muscle PK (9) indicated that Lys-269 (Lys-240 in the YPK numbering sequence) can serve as the putative proton donor because it was positioned close to the methyl carbon of the bound pyruvate. The ⑀-amino group of ...

The Functional Glycosyltransferase Signature Sequence of the Human β1,3-Glucuronosyltransferase Is a XDD Motif

“…Altogether, these data show that divalent ions are essential for GlcAT-I activity as well as for substrate binding, but do not provide information on the activation mechanism. Kinetic analyses combined with site-directed mutagenesis have been successfully used to assess metal effects in enzyme activation (32,33). We thus initiated kinetic studies of Mn 2ϩ activation of wild-type and mutant GlcAT-I.…”

Monitoring Active Site Alterations upon Mutation of Yeast Pyruvate Kinase Using 205Tl+ NMR

“…Each mutant enzyme has different kinetic characteristics. The results led to the conclusion that a molecule of bound water at the active site serves as the proton donor (6). In an effort to characterize further the effect of these mutations at the active site, the interaction between the enzyme-bound divalent activator (Mn 2ϩ ) and the monovalent activator (Tl ϩ ) was investigated in each of the mutants by appropriate NMR studies.…”

“…The measurement of 1/T 1M values at 346 MHz for wild type YPK and the T298S mutant enzyme-Tl ϩ -PEP and enzyme-Tl ϩ -PEP-FBP complexes indicates that there is a frequency dependence for 1/T 1M , based on Equation 6 and that the measured values reflect relaxation (Table III). There is no frequency dependence for the 1/pT 2p values for these complexes (Table III).…”

“…All three Thr-298 mutants of YPK, T298S, T298C and T298A, showed altered k cat and k cat /K m,PEP values and altered kinetic cooperativity with PEP relative to wild type PK (6). It is possible that these alterations in kinetic parameters of the YPK Thr-298 mutants may, in part, be explained by structural changes at the active site as a result of the single amino acid mutations.…”

“…1). The role of Thr-298 in the YPK-catalyzed reaction has been addressed by mutation of this residue to serine, alanine (6), and to cysteine. 2 Studies of these mutants suggest that Thr-298 is not the proton donor to the enolate of pyruvate but that enzyme-bound water serves this function.…”