Stereochemistry of ketonization of enolpyruvate by pyruvate kinase. Evidence for its role as an intermediate

Kuo, Donald J.; Rose, Irwin A.

doi:10.1021/ja00487a084

Cited by 28 publications

(21 citation statements)

References 8 publications

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“…It has been noted that the amino group of Lys-265 is sufficiently close to the active site to be involved in the catalytic ketonization of bound enolpyruvate (Muirhead et al, 1986). Such a lysine has been suggested by Tipton et al (1989) as the group having pK = 8.3 (Dougherty & Cleland, 1985) that is responsible for the stereospecific ketonization of enolpyruvate (Kuo & Rose, 1978). An intruiguing hypothesis is that the observed weakly coupled outer shell proton may originate from Lys-265 and be hydrogen bonded to the oxalate inhibitor in the location which it occupies when it provides a proton to enolpyruvate.…”

Section: Proton Endor Resultsmentioning

confidence: 98%

Electron nuclear double resonance study of the manganese(2+) environs in the oxalate-ATP complex of pyruvate kinase

Tan

Poyner²,

Reed³

et al. 1993

Biochemistry

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Electron nuclear double resonance (ENDOR) and the related pulse technique of pulse field sweep EPR (PFSEPR) were used to probe the site I environment of Mn2+ in the oxalate-ATP complex of pyruvate kinase. Assignment of features and an estimate of hyperfine couplings have shown proximity of protons to the metal ions through their dipolar interaction and proximity of 31P and 17O because of a contact interaction from direct Mn(2+)-ligand covalent spin transfer. Since Mn2+ is a spin5/2 ion whose Ms = +/- 1/2, +/- 3/2, and +/- 5/2 electron spin states can all contribute to EPR and ENDOR, we have developed experimental and theoretical strategies for elucidating hyperfine couplings to the Mn2+ electron spin states. Solvent-exchangeable proton ENDOR features were evident with couplings very similar to the hyperfine couplings of H2O in [Mn(H2O)6]2+. ENDOR of exchangeable, more distant protons originated from a dipolar coupling such as could be expected from protons residing 5.5 A from Mn2+ and hydrogen-bonded to a nonliganding oxygen or nitrogen. Nonexchangeable proton ENDOR features indicated dipolar coupling to proton(s) from the protein residing at approximately 4.5 A from the Mn2+. The approximately 4-MHz 31P phosphate hyperfine couplings in Mn(II)-nucleotide models and in pyruvate kinase were similar, but a detailed ENDOR and PFSEPR comparison revealed that the hyperfine coupling to the ATP gamma-phosphate in pyruvate kinase was approximately 10% less than coupling to phosphates of Mn(II)-nucleotides. [In pyruvate kinase only the gamma-phosphate has been shown to bind to Mn2+ at site I (Lodato & Reed, 1987).](ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Proton Endor Resultsmentioning

confidence: 98%

Electron nuclear double resonance study of the manganese(2+) environs in the oxalate-ATP complex of pyruvate kinase

Tan

Poyner²,

Reed³

et al. 1993

Biochemistry

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“…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).…”

mentioning

confidence: 99%

“…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).…”

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confidence: 99%

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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“…35 Our longstanding belief in enolpyruvate as the intermediate in the pyruvate kinase reaction 37,38 was confirmed by chemical analysis of the active enzyme. 39 Synthesis of enolpyruvate by treating D, T-PEP with phosphatase made it possible to determine the stereochemistry of pyruvate kinase 40 and its role in transcarboxylase. 41 In a surprise result, the Schiffs bases of dihydroxyacetone-P and of fructose-1,6 P2 were found to precipitate with the protein of muscle aldolase in cold acid.…”

Section: Cdd: and When Did You Move To The Fox Chase And Begin The Comentioning

confidence: 99%

Early work on the ubiquitin proteasome system, an interview with Irwin Rose

Rose

2005

Cell Death Differ

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Stereochemistry of ketonization of enolpyruvate by pyruvate kinase. Evidence for its role as an intermediate

Cited by 28 publications

References 8 publications

Electron nuclear double resonance study of the manganese(2+) environs in the oxalate-ATP complex of pyruvate kinase

Electron nuclear double resonance study of the manganese(2+) environs in the oxalate-ATP complex of pyruvate kinase

The Proton Transfer Step Catalyzed by Yeast Pyruvate Kinase

Early work on the ubiquitin proteasome system, an interview with Irwin Rose

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