Stereochemical course of phosphokinases. The use of adenosine [.gamma.-(S)-16O,17O,18O]triphosphate and the mechanistic consequences for the reactions catalyzed by glycerol kinase, hexokinase, pyruvate kinase, and acetate kinase

Aluminum fluoride has become an important tool for investigating the mechanism of phosphoryl transfer, an essential reaction that controls a host of vital cell functions. Planar AlF 3 or AlF 4 ؊ molecules are proposed to mimic the phosphoryl group in the catalytic transition state. Acetate kinase catalyzes phosphoryl transfer of the ATP ␥-phosphate to acetate. Here we describe the inhibition of acetate kinase from Methanosarcina thermophila by preincubation with MgCl 2 , ADP, AlCl 3 , NaF, and acetate. Preincubation with butyrate in place of acetate did not significantly inhibit the enzyme. Several NTPs can substitute for ATP in the reaction, and the corresponding NDPs, in conjunction with MgCl 2 , AlCl 3 , NaF, and acetate, inhibit acetate kinase activity. Fluorescence quenching experiments indicated an increase in binding affinity of acetate kinase for MgADP in the presence of AlCl 3 , NaF, and acetate. These and other characteristics of the inhibition indicate that the transition state analog, MgADP-aluminum fluoride-acetate, forms an abortive complex in the active site. The protection from inhibition by a non-hydrolyzable ATP analog or acetylphosphate, in conjunction with the strict dependence of inhibition on the presence of both ADP and acetate, supports a direct in-line mechanism for acetate kinase.

Section: Discussionmentioning

confidence: 98%

Evidence for a Transition State Analog, MgADP-Aluminum Fluoride-Acetate, in Acetate Kinase from Methanosarcina thermophila

Miles

Gorrell

Ferry

2002

“…Additionally, the discovery that the E. coli acetate kinase is able to phosphorylate enzyme I of the phosphotransferase system (12) and CheY (13) in vitro indicates the phosphoenzyme functions in sugar transport. Later investigations reported inversion of the stereochemistry about the phosphorous (14) and isotope exchange kinetics inconsistent with the covalent kinase mechanism (15) and supporting a direct in-line phosphoryl transfer. More recently, the acetate kinase from M. thermophila was shown to be inhibited by components of a putative transition state analogue ADP-AlF x -acetate (16) in which the AlF x is proposed to mimic the meta-phosphate in a direct phosphoryl transfer mechanism.…”

mentioning

confidence: 99%

Structural and Kinetic Analyses of Arginine Residues in the Active Site of the Acetate Kinase from Methanosarcina thermophila

Gorrell¹,

Lawrence

Ferry

2005

Phosphoryl transfer is a key reaction in numerous biological processes, playing roles in signaling mechanisms, energy transfer, and energy storage in both eukaryotic and prokaryotic cells (1). One of the earliest phosphoryl transfers identified was the phosphorylation of acetate by ATP to form acetyl phosphate (AcP) 1 and ADP, described in 1944 by Lippman (2). This reversible reaction is catalyzed by acetate kinase, which is widely distributed among anaerobic prokaryotes playing a central role in energy-yielding metabolism by synthesizing ATP from acetyl phosphate generated in fermentation pathways. The enzyme also plays an essential role in the fermentation of acetate to methane, which accounts for most of the one billion metric tons of methane produced annually from the decomposition of organic matter by anaerobic microbial consortia (3). In Methanosarcina thermophila, acetate kinase catalyzes the first step in the pathway by activating acetate to acetyl phosphate prior to transfer of the acetyl moiety to CoA catalyzed by phosphotransacetylase (4, 5). In later steps of the pathway, the acetyl moiety is further metabolized to methane and carbon dioxide (6). Although acetate kinase was one of the first enzymes to be investigated mechanistically, details remain elusive; indeed, the first crystal structure was obtained only recently for the M. thermophila enzyme, identifying acetate kinase as a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) structural superfamily and the best candidate for the common ancestor of this family (7). The earliest kinetic studies of the enzyme from Escherichia coli suggested a ping-pong mechanism (8), and evidence for a covalent phosphoryl intermediate supported this mechanism (9, 10); however, it was later shown that the phosphoryl-enzyme complex is not kinetically competent (11). Additionally, the discovery that the E. coli acetate kinase is able to phosphorylate enzyme I of the phosphotransferase system (12) and CheY (13) in vitro indicates the phosphoenzyme functions in sugar transport. Later investigations reported inversion of the stereochemistry about the phosphorous (14) and isotope exchange kinetics inconsistent with the covalent kinase mechanism (15) and supporting a direct in-line phosphoryl transfer. More recently, the acetate kinase from M. thermophila was shown to be inhibited by components of a putative transition state analogue ADP-AlF x -acetate (16) in which the AlF x is proposed to mimic the meta-phosphate in a direct phosphoryl transfer mechanism. No structural evidence for either the covalent or in-line mechanism has been reported previously.Access to the crystal structure (7) and production of the M. thermophila acetate kinase in E. coli (17) have allowed experimental approaches not previously employed to investigate the catalytic mechanism of this enzyme. The structure of the homodimeric acetate kinase co-crystallized with ATP (the ATP-AK structure) reveals ADP in a cleft with contacts that are conserved in the nucleotide binding sites of other ASKHA f...

“…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 .…”

mentioning

confidence: 99%

“…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)(3)(4).…”

mentioning

confidence: 99%

The Proton Transfer Step Catalyzed by Yeast Pyruvate Kinase

Susan‐Resiga

Nowak

2003

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 ...