Pyruvate formate-lyase reaction

Pyruvate formate-lyase reaction. Anaerobic cleavage of pyruvate to acetyl-CoA and formate (Eq. 15-37) is essential to the energy economy of many cells, including those of E. coli. No external oxidant is needed, and the reaction does not require lipoic acid. 

The details of the process and the oxidation-reduction balance can be pictured as in Eq. 17-25. Pyruvate is cleaved by the pyruvate formate-lyase reaction (Eq. 15-37) to acetyl-CoA and formic acid. Half of the acetyl-CoA is cleaved to acetate via acetyl-P with generation of ATP, while the other half is reduced in two steps to ethanol using the two molecules of NADH produced in the initial oxidation of triose phosphate (Eq. 17-25). The overall energy yield is three molecules of ATP per glucose. The "efficiency" is thus (3 x 34.5)... 

Use the pyruvate formate-lyase reaction. What yield of ATP do you expect per molecule of glucose fermented ... 

When grown in alkaline media, certain species of lactic acid bacteria decrease production of LDH, resulting in increased formation of formate, acetate, and ethanol as end products. This phenomenon has been observed in S. faecalis subsp. liquefaciens (Gunsalus and Niven 1942), Streptococcus durans, S. thermophilus, (Platt and Foster 1958), and Lactobacillus bulgaricus (Rhee and Pack 1980). Data of Rhee and Pack (1980) indicate that a phosphoroclastic split of pyruvate occurs under alkaline conditions to yield ATP. The enzymes involved in this reaction (pyruvate formate-lyase and acetate kinase) require alkaline conditions for optimum activity. A shift from homo- to heterofermentation because of increased pH has not been observed for Group N streptococci. 

The a-oxoacid dehydrogenases yield CoA derivatives which may enter biosynthetic reactions. Alternatively, the acyl-CoA compounds may be cleaved with generation of ATP. The pyruvate formate-lyase system also operates as part of an ATP-generating system for anaerobic organisms, for example, in the "mixed acid fermentation" of enterobacteria such as E. coli (Chapter 17). These two reactions, which are compared in Fig. 15-16, constitute an important pair of processes both of which accomplish substrate-level phosphorylation. They should be compared with the previously considered examples of substrate level phosphorylation depicted in Eq. 14-23 and Fig. 15-16. 

Pyruvaterferredoxin oxidoreductase 799 Pyruvate formate-lyase 875 reaction 800, 801 Pyruvate kinase 656, 706... 

The best-studied enzymes to date that contain glycyl radicals are pyruvate formate-lyase (PFL) and a ribonucleoside triphosphate reductase (ARR), both isolated from anaerobically growing E. coli. These enzymes play central roles in the anaerobic metabolism of the bacterium. The first catalyzes the reversible formation of acetyl-CoA and formate from pyruvate and coenzyme A, while the second is responsible for synthesizing the deoxyribonucleotide monomers of the polymer DNA. It is intriguing to note that formate, a product of the PFL reaction, is a substrate for the ARR. It supplies the reducing equivalents needed for each round of deoxynucleotide synthesis. ... 

We have in the present chapter shown results from theoretical model system studies of the catalytic reaction mechanisms of three radical enzymes Galatose oxidase. Pyruvate formate-lyase and Ribonucleotide reductase. It is concluded that small models of the key parts of the active sites in combination with the DPT hybrid functional B3LYP and large basis sets provides a good description of the catalytic machineries, with low barriers for the rate determining steps and moderate overall exothermicity. The models employed are furthermore able to reproduce all the observed features in terms of spin distributions and reactive intermediates. 

In Pyruvate formate-lyase, the active site contains a stable glycyl radical and two catalyticaUy active cysteines. Two different proposed reaction mechanisms were tested, and it was concluded that the mechanism as suggested... 

Pyruvate formate lyase that catalyzes the conversion of pyruvate to formate and acetyl CoA is a key enzyme in the anaerobic degradation of carbohydrates in some enteric bacteria. Using an enzyme selectively 13C-labeled with glycine, it was shown by EPR that the reaction involves production of a free radical at C-2 of glycine (Wagner et al. 1992). This was confirmed by destruction of the radical with 02, and determination of part of the structure of the small protein which contained an oxalyl residue originating from gly-734. 

Several enzymes utilize thiyl radicals for substrate conversion. In the ribonucleotide reductase (RNR) class III, pyruvate formate lyase and benzylsuccinate synthase, cysteine thiyl radicals are generated via hydrogen transfer from cysteine to glycine radicals (Reaction (3.25)) [3, 24-27, 48-52]. 

Pyruvate dehydrogenase and pyruvate-formate lyase are enzymes that are endogenous to E. coli and which process pyruvate into acetyl coenzyme A. Acetyl phosphate is then produced from acetyl coenzyme A by phosphotransacetylase, also endogenous to E. coli. In this way, pyruvate can be processed in E. coli to yield acetyl phosphate, a phosphate donor (3). The activities of these pathways in E. coli lysate were tested [7] by supplying a batch-mode reaction with pyruvate in the absence of an alternative secondary energy store. 

Pyruvate formate-lyase (EC 2.3.1.54 formate acetyltransferase PEL) catalyzes the key reaction in anaerobic glucose metabolism in bacteria, the coenzyme A-dependent dismutation of pyruvate into acetyl-CoA and formate. The reaction, first reported by Werkman and co-workers in the early 1940s (169, 170), was described as the phosphoroclastic cleavage of pyruvate because acetyl phosphate was detected as a product. Following the discovery of CoA and the elucidation of its role in acetyl transfer reactions (77/, 772), the intermediacy of acetyl-CoA in pyruvate dismutation was realized the overall reaction catalyzed by PFL is generally described by two half-reactions (Scheme 37). 

Scheme 37. Half-reactions of pyruvate formate-lyase 187).

The inhibition of pyruvate formate-lyase by hypophosphite was first observed by Novelli in work on the CoA-independent carboxylate exchange reaction between pyruvate and formate (186). In a more detailed study by Knappe et al., time-dependent inactivation is observed to occur with concomitant loss of the enzyme free radical EPR signal (180). The inactivation kinetics are first order and the rate of inactivation is accelerated when the enzyme is in the acetylated form. Furthermore, inactivation by [ HJhypophosphite leads to the stoichiometric release of tritium to H2O, and treatment of PFL with [ P]hypophosphite produces an alkali-labile radiolabel that is covalently bound to the inactive enzyme (180). 

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