Substrate proton of the pyruvate kinase reaction

Rose, Irwin A.; Kuo, Donald J.

doi:10.1021/bi00451a005

Cited by 22 publications

(31 citation statements)

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“…On the other hand, only a relatively small amount of solvent hydrogen is found in the methyl groups of the leucine moiety. The biosynthesis of leucine from two molecules of pyruvate and one molecule of acetyl-CoA involves the introduction of one solvent hydrogen atom into each of the leucine methyl groups because of the transformation of phosphoenol pyruvate to pyruvate (11)(12)(13). Additional solvent hydrogen can be introduced into pyruvate by spontaneous proton exchange because of the relatively high acidity of the pyruvate methyl group.…”

Section: Resultsmentioning

confidence: 99%

Biosynthetic Origin of Hydrogen Atoms in the Lipase Inhibitor Lipstatin

Goese

Eisenreich

Kupfer

et al. 2000

Journal of Biological Chemistry

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Section: Resultsmentioning

confidence: 99%

Biosynthetic Origin of Hydrogen Atoms in the Lipase Inhibitor Lipstatin

Goese

Eisenreich

Kupfer

et al. 2000

Journal of Biological Chemistry

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“…No other side chain is within less than 5 A of His 373 N'. Thus, the topography at the active site does not appear to favor the existence of the kind of multiple proton pool such as was described for some enzymes that catalyze proton abstraction from carbon (Kuo & Rose, 1987;Rose & Kuo, 1989;Rose et al, 1990). The presence of a number of fixed water molecules at the active site of subunit S1 (no ligand present) might at first sight suggest the possibility of a water-mediated proton transfer.…”

Section: Extreme P K Displacements At Enzyme Active Sites 543mentioning

confidence: 93%

Extreme pK_a displacements at the active sites of FMN‐dependent α‐hydroxy acid‐oxidizing enzymes

Lederer

1992

Protein Science

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Flavocytochrome b2 (or L-lactate dehydrogenase) from baker's yeast is thought to operate by the initial formation of a carbanion, as do the evolutionarily related alpha-hydroxy acid-oxidizing FMN-dependent oxidases. Previous work has shown that, in the active site of the unligated reduced flavocytochrome b2, the group that has captured the substrate alpha-proton has a high pKapp, calculated to lie around 15 through the use of Eigen's equation. A detailed inspection of the now known three-dimensional structure of the enzyme leads to the conclusion that the high pKa belongs to His 373, an active site group that plays the role of general base in the forward reaction and of general acid in the reverse direction. Moreover, consideration of the kinetics of proton transfer during the catalytic cycle suggests that the pKa of the reduced FMN N5 position should be lowered by several pH units compared to its pKa of 20 or more when free. The features of the three-dimensional structure possibly responsible for these pK shifts are analyzed; they are proposed to consist of a network of hydrogen bonds with the solvent and of a mutual electrostatic stabilization of anionic reduced flavin and the imidazolium ion. Finally, it is suggested that similar pK shifts affect the active sites of the alpha-hydroxy acid-oxidizing flavooxidases, which are homologous to flavocytochrome b2. The functional significance of these pK shifts in terms of catalysis and semiquinone stabilization is discussed.

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“…The ⑀-amino group of Lys-269 as the possible candidate for the acid/base catalyst in PK was supported by the measurement of multiple protons that are incorporated into pyruvate with rabbit muscle pyruvate kinase (10). Subsequent refined x-ray crystal structures of the yeast enzyme with bound phosphoglycolate (11) and of the rabbit muscle enzyme complexed with pyruvate (12) demonstrated that Lys-240 is at the 2-re face of the double bond of the enolate and is in apparent contact with the phosphoryl group of PEP.…”

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confidence: 98%

The Proton Transfer Step Catalyzed by Yeast Pyruvate Kinase

Susan‐Resiga

Nowak

2003

Journal of Biological Chemistry

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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 ...

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Substrate proton of the pyruvate kinase reaction

Cited by 22 publications

References 30 publications

Biosynthetic Origin of Hydrogen Atoms in the Lipase Inhibitor Lipstatin

Biosynthetic Origin of Hydrogen Atoms in the Lipase Inhibitor Lipstatin

Extreme pK_a displacements at the active sites of FMN‐dependent α‐hydroxy acid‐oxidizing enzymes

The Proton Transfer Step Catalyzed by Yeast Pyruvate Kinase

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Substrate proton of the pyruvate kinase reaction

Cited by 22 publications

References 30 publications

Biosynthetic Origin of Hydrogen Atoms in the Lipase Inhibitor Lipstatin

Biosynthetic Origin of Hydrogen Atoms in the Lipase Inhibitor Lipstatin

Extreme pKa displacements at the active sites of FMN‐dependent α‐hydroxy acid‐oxidizing enzymes

The Proton Transfer Step Catalyzed by Yeast Pyruvate Kinase

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Product

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Extreme pK_a displacements at the active sites of FMN‐dependent α‐hydroxy acid‐oxidizing enzymes