Physical, chemical, and enzymological characterization of enolpyruvate

Kuo, Donald J.; O'Connell, Edward L.; Rose, Irwin A.

doi:10.1021/ja00511a037

Cited by 39 publications

(38 citation statements)

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“…The possibility of kinetic factors in the cooperative response of T298S was investigated by measuring the velocity response to variable [PEP] in a temperature range from 8 to 40°C. 4 The apparent Hill coefficients for PEP do not change significantly over the temperature range studied. This suggests that if there is a kinetic step (or steps) that attenuates the cooperative response of PEP by the Mn 2ϩ -activated enzyme in the absence of Fru-6-P 2 , then this step is not temperature-sensitive.…”

Section: Discussionmentioning

confidence: 88%

“…This suggests that the active site Thr-298 does not directly affect the phosphoryl transfer step of the PK-catalyzed reaction. There is evidence 4 that the mutations of Thr-298 elicit conformational changes of bound PEP at the catalytic site where phosphoryl transfer does occur, although these changes do not extend into the nucleotide-binding site. The kinetic studies of glycolate kinase activity indicate that the interaction of ATP with the enzyme is unaffected by mutation of Thr-298.…”

Section: Discussionmentioning

confidence: 99%

“…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)(3)(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).…”

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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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Section: Discussionmentioning

confidence: 88%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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The Proton Transfer Step Catalyzed by Yeast Pyruvate Kinase

Susan‐Resiga

Nowak

2003

Journal of Biological Chemistry

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“…Pyruvate kinase (PK 3 ; EC 2.7.1.40), a pacemaker of the glycolytic pathway, catalyzes irreversibly the transphosphorylation from P-enolpyruvate to ADP, generating pyruvate and ATP (1,2). There are four different isozymes (L, R, M 1 , and M 2 ) in mammalian tissues, which differ in their regulatory properties.…”

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confidence: 99%

“…The M 1 isozyme is regulated by neither P-enolpyruvate nor Fru-1,6-P 2 because of its intrinsic active conformation in the R-state (5,6). Under unfavorable conditions such as hypoxia and lack of glucose supply, the anaerobic tissues and tumor cells rely heavily on PKM 2 for ATP production (7). Therefore, stringent control of PK activity is of great importance not only for cell metabolism but also for tumorigenic proliferation.…”

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confidence: 99%

Differential Behavior of Missense Mutations in the Intersubunit Contact Domain of the Human Pyruvate Kinase M2 Isozyme

Akhtar

Gupta

Koul

et al. 2009

Journal of Biological Chemistry

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In this study, we attempted to understand the mechanism of regulation of the activity and allosteric behavior of the pyruvate kinase M 2 enzyme and two of its missense mutations, H391Y and K422R, found in cells from Bloom syndrome patients, prone to develop cancer. Results show that despite the presence of mutations in the intersubunit contact domain, the K422R and H391Y mutant proteins maintained their homotetrameric structure, similar to the wild-type protein, but showed a loss of activity of 75 and 20%, respectively. Interestingly, H391Y showed a 6-fold increase in affinity for its substrate phosphoenolpyruvate and behaved like a non-allosteric protein with compromised cooperative binding. However, the affinity for phosphoenolpyruvate was lost significantly in K422R. Unlike K422R, H391Y showed enhanced thermal stability, stability over a range of pH values, a lesser effect of the allosteric inhibitor Phe, and resistance toward structural alteration upon binding of the activator (fructose 1,6-bisphosphate) and inhibitor (Phe). Both mutants showed a slight shift in the pH optimum from 7.4 to 7.0. Although this study signifies the importance of conserved amino acid residues in long-range communications between the subunits of multimeric proteins, the altered behavior of mutants is suggestive of their probable role in tumor-promoting growth and metabolism in Bloom syndrome patients with defective pyruvate kinase M 2 .Pyruvate kinase (PK 3 ; EC 2.7.1.40), a pacemaker of the glycolytic pathway, catalyzes irreversibly the transphosphorylation from P-enolpyruvate to ADP, generating pyruvate and ATP (1, 2). There are four different isozymes (L, R, M 1 , and M 2 ) in mammalian tissues, which differ in their regulatory properties. These isozymes are allosteric in nature with the exception of the M 1 form, present in skeletal muscle and brain (3-7). PKM 2 is a ubiquitous prototype enzyme present in all tissues during the embryonic stage and is gradually replaced by other isozymic forms in specific tissues during development. The M 2 , L, and R isozymes show homotropic cooperative activation with P-enolpyruvate and heterotropic cooperative activation with Fru-1,6-P 2 (8 -10). The M 1 isozyme is regulated by neither P-enolpyruvate nor Fru-1,6-P 2 because of its intrinsic active conformation in the R-state (5, 6). Under unfavorable conditions such as hypoxia and lack of glucose supply, the anaerobic tissues and tumor cells rely heavily on PKM 2 for ATP production (7). Therefore, stringent control of PK activity is of great importance not only for cell metabolism but also for tumorigenic proliferation.The M 1 and M 2 isozymes are produced from a single gene locus by mutually exclusive alternative splicing (11)(12)(13)(14). In the human M 1 and M 2 isozymes, the exon that is exchanged because of alternative splicing encodes 56 amino acids, in which a total of 22 amino acids differ within a length of 45 residues. The residues located in this region form the major intersubunit contact domain (8). The distinguishable kinetic pro...

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