Irreversible Inactivation of Pyruvate Decarboxylase in the Presence of Substrate and an Oxidant. An Example of Paracatalytic Enzyme Inactivation

In Krebs-Ringer phosphate buffer, the rate of formation of methylglyoxal from glycerone phosphate and glyceraldehyde 3-phosphate was first order with respect to the triose phosphate with rates constant values of 1.94 2 0.02X10-5 s-' (n = 18) and 1.54 2 0.02X10-4 s-' (n = 18) at 37"C, respectively. The rate of formation of methylglyoxal from glycerone phosphate and glyceraldehyde 3-phosphate in the presence of red blood cell lysate was not significantly different from the nonenzymatic value ( P > 0.05). Methylglyoxal formation from glycerone phosphate was increased in the presence of triose phosphate isomerase but this may be due to the faster non-enzymatic formation from the glyceraldehyde 3-phosphate isomerisation product. For red blood cells in vitro, the predicted non-enzymatic rate of formation of methylglyoxal from glycerone phosphate and glyceraldehyde 3-phosphate may account for the metabolic flux through the glyoxalase system. The reactivity of glycerone phosphate and glyceraldehyde 3-phosphate towards the non-enzymatic formation of methylglyoxal under physiological conditions suggests that methylglyoxal formation is unavoidable from the Embden-Meyerhof pathway.Methylglyoxal (2-oxopropanal) is a metabolite found widespread throughout biological life (Thornalley, 1990 ;Murata et al., 1989;Ohmori et al., 1989;Steinberg and Kaplan, 1984). It was initially thought to be involved in mainstream glycolysis but the discovery of phosphorylated glycolytic intermediates led to its demise as a metabolite of major importance. It has been most widely investigated with respect to the glyoxalase system which catalyses the conversion of methylglyoxal to D-lactate via the intermediate S-(D1actoyl)glutathione (Thornalley, 1990) but it is now receiving renewed interest as a substrate for aldose reductase (Vander Jagt et al., 1992). The toxicity of high doses of methylglyoxal to biological tissue prompted Albert Szent-Gyorgyi to suggest that methylglyoxal was a 'growth-inhibiting factor' or 'retine', and the glyoxalase system a 'growth-promoting factor' or 'promine', and the conflict between these two factors was involved in control of cell growth (Szent-Gyorgyi, 1977). This hypothesis has been superseded by the discovery of oncogenes and peptide factors (Bradshaw and Prentice, 1987), although recent evidence suggests that methylglyoxal, by conversion to S-(D-lactoyl)glutathione, may influence cell Correspondence to P. J. Thornalley,

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“…dium iodoacetate (1 mM) to inhibit glyceraldehyde-3-phosphate dehydrogenase (Cogoli-Greuter and Christen, 1981).…”

Section: Non-enzymatic Formation Of Methylglyoxal From Glycerone Phosmentioning

confidence: 99%

The formation of methylglyoxal from triose phosphates

Phillips

Thornalley

1993

European Journal of Biochemistry

508

318

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“…POX Catalysis in the Presence of DCPIP-DCPIP can act as an artificial electron acceptor in ThDP-dependent enzymes (6,7,13), and, e.g. in pyruvate decarboxylase from yeast (EC 4.1.1.1.…”

mentioning

confidence: 99%

Activation of Thiamin Diphosphate and FAD in the Phosphatedependent Pyruvate Oxidase fromLactobacillus plantarum

Tittmann¹,

Proske²,

Spinka³

et al. 1998

Journal of Biological Chemistry

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The phosphate-and oxygen-dependent pyruvate oxidase from Lactobacillus plantarum is a homotetrameric enzyme that binds 1 FAD and 1 thiamine diphosphate per subunit. A kinetic analysis of the partial reactions in the overall oxidative conversion of pyruvate to acetyl phosphate and CO 2 shows an indirect activation of the thiamine diphosphate by FAD that is mediated by the protein moiety. The rate constant of the initial step, the deprotonation of C2-H of thiamine diphosphate, increases 10-fold in the binary apoenzyme-thiamine diphosphate complex to 10 ؊2 s ؊1 . Acceleration of this step beyond the observed overall catalytic rate constant to 20 s ؊1 requires enzyme-bound FAD. FAD appears to bind in a two-step mechanism. The primarily bound form allows formation of hydroxyethylthiamine diphosphate but not the transfer of electrons from this intermediate to O 2 . This intermediate form can be mimicked using 5-deaza-FAD, which is inactive toward O 2 but active in an assay using 2,6-dichlorophenolindophenol as electron acceptor. This analogue also promotes the rate constant of C2-H dissociation of thiamine diphosphate in pyruvate oxidase beyond the overall enzyme turnover. Formation of the catalytically competent FAD-thiamine-pyruvate oxidase ternary complex requires a second step, which was detected at low temperature. Pyruvate oxidase (POX)1 from Lactobacillus plantarum (EC 1.2.3.3) is a homotetramer. Each subunit (mass, 65.5 kDa) binds one FAD and one thiamine diphosphate (ThDP) in the presence of Mn 2ϩ or Mg 2ϩ . In the presence of oxygen and phosphate, the POX holoenzyme catalyzes the oxidative decarboxylation of pyruvate (1-3) according to the equationIn the presence of Mn 2ϩ , both coenzymes FAD and ThDP can form binary complexes with the apoenzyme that are enzymatically inactive in the native overall oxidation reaction (4).The important catalytic steps in the POX reaction are: step 1, deprotonation of C2-H of ThDP, the initial step shared by all ThDP dependent enzymes; step 2, binding of pyruvate to the C2 atom of enzyme-bound ThDP; step 3, decarboxylation of pyruvate to hydroxyethyl-ThDP; step 4, oxidation of hydroxyethylThDP by FAD; and step 5, reoxidation of reduced FAD by oxygen. To study the role of both coenzymes in POX catalysis, we have used four different approaches that selectively investigate some of the individual steps enumerated above: (a) In the initial reaction (step 1), the deprotonation of the C2-atom of the ThDP in POX holoenzyme as well as in the binary ThDP complex was studied using 1 H NMR by following the C2-H/D exchange (5). (b) DCPIP is known to accept electrons from hydroxyethyl-ThDP in other ThDP-dependent enzymes (6, 7). Here it was used as an artificial acceptor to assess the mode of electron transfer to O 2 . Two alternative donor loci have to be considered, the ThDP adduct itself or FADH 2 , to which redox equivalents could be transferred from ThDP. We have also used this assay with POX in which normal FAD was substituted with the analogue 5-deaza FAD (5-dFAD), which is essential...

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“…For example, fructose-l,6-bisphosphate aldolase (a Schiff-base-forming class I aldolase) undergoes paracatalytic modification in the presence of fructose 1,6-bisphosphate and hexacyanoferrate(1II) (Scheme 1). Similar paracatalytic modification accompanying the oxidation of an enzyme-substrate carbanion intermediate also occurs with zincdependent class II fructose-1,6-bisphosphate aldolase from yeast, transaldolase, transketolase [6], pyruvate decarboxylase [8] and ribulose-l,5-bisphosphate carboxylase-oxygenase [9]. Paracatalytic modification is observed only with the specific substrate that forms the enzyme-substrate carbanion intermediate but does not depend on a specific oxidant.…”

Section: N-lys146mentioning

confidence: 96%

Paracatalytic self‐inactivation of fructose‐1,6‐bisphosphate aldolase

Gupta¹,

Hollenstein²,

Kochhar³

et al. 1993

European Journal of Biochemistry

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Oxidation of enzyme‐substrate carbanion intermediates by extrinsic oxidants may result in irreversible paracatalytic inactivation of certain enzymes. In paracatalytically modified fructose‐1,6‐bisphosphate aldolase from rabbit muscle the polypeptide chain had been found to be crosslinked at active‐site Lys229 (Schiff base forming with substrate) and Lys146 by a phosphorylated three‐carbon moiety [Lubini, D. G. E. and Christen, P. (1979) Proc. Natl Acad. Sci. USA 76, 2527–2531]. In the present study, the structure of this crosslink was elucidated by instrumental analysis. Aldolase was paracatalytically modified in the presence of fructose 1,6‐bisphosphate and hexacyanoferrate(III). The completely inactivated enzyme was digested with pronase. The crosslinked peptide was isolated by gel filtration and reverse‐phase HPLC. Mass spectroscopy, 1H‐ and 13C‐NMR showed that a derivative of dihydroxyacetone phosphate forms an amidine with the ɛ‐amino groups of the two lysine residues:

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Irreversible Inactivation of Pyruvate Decarboxylase in the Presence of Substrate and an Oxidant. An Example of Paracatalytic Enzyme Inactivation

Cited by 16 publications

References 27 publications

The formation of methylglyoxal from triose phosphates

The formation of methylglyoxal from triose phosphates

Activation of Thiamin Diphosphate and FAD in the Phosphatedependent Pyruvate Oxidase fromLactobacillus plantarum

Paracatalytic self‐inactivation of fructose‐1,6‐bisphosphate aldolase

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