Pyruvate oxidase active site

Figure 14-2 (A) Stereoscopic view of the active site of pyruvate oxidase from the bacterium Lactobacillus plantarium showing the thiamin diphosphate as well as the flavin part of the bound FAD. The planar structure of the part of the intermediate enamine that arises from pyruvate is shown by dotted lines. Only some residues that may be important for catalysis are displayed G35 , S36 , E59 , H89 , F12T, Q122 , R264, F479, and E483. Courtesy of Georg E. Schulz.119 (B) Simplified view with some atoms labeled and some side chains omitted. The atoms of the hypothetical enamine that are formed from pyruvate, by decarboxylation, are shown in green.

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. 

By considering the above-mentioned solution studies and the refined three-dimensional structure of the S. cerevisiae flavocytochrome 62 active site, Lederer and Mathews proposed a scheme for the reverse reaction (the reduction of pyruvate) (39). They did not discuss how the transfer of electrons took place except to say that the structure did not rule out the possibility of a covalent intermediate (39). Ghisla and Massey (116) considered the anionic flavin N5 to be too close to the pyruvate carbonyl (3.7 A) without the formation of a covalent adduct taking place. Covalent intermediates between substrate and flavin have been observed for lactate oxidase (117, 118) and o-amino acid... 

Ethanol accumulated in maturing citrus fruit as the end product of pyruvate decarboxylation. Conditions that promote this reaction include low and high CC, and ethylene levels. Maturation increased the levels of PDC and ADH and increased the NADH to NAD ratio. The higher redox ratio could slow the PDH reaction which competes with PDC for pyruvate. Development of the alternative oxidase activity when ethanol began to accumulate suggests that membrane function was modified which affected rates of various metabolic pathways. The lower phosphorylation efficiency of the alternative oxidase compared to the cytochrome pathway (22) could affect numerous metabolic activities including decarboxylation of pyruvate. Also, membrane transport of pyruvate and cofactors could be altered in mitochondria containing fewer phosphorylation sites (26). 

On the basis of the crystal structure of a Bacillus stearothermophilus pyruvate dehydrogenase subcomplex formed between the heterotetrameric El and the peripheral subunit binding domain of E2 with an evident stmctural dissymmetry of the two active sites, a direct active center communication via an acidic proton tunnel has been proposed (Frank et ak, 2004). According to this, one active site is in a closed state with an activated cofactor even before a substrate molecule is engaged, whereas the activation of the second active site is coupled to decarboxylation in the first site. Our own kinetic NMR studies on human PDH El (unpublished) support the model suggested, but similar studies on related thiamin enzymes, such as pyruvate decarboxylase, transketolase or pyruvate oxidase reveal that half-of-the-sites reactivity is a unique feature of ketoacid dehydrogenases. In line with this. X-ray crystallography studies on intermediates in transketolase catalysis indicated an active site occupancy close to unity in both active sites (Fiedler et al., 2002 and G. Schneider, personal communication). 

ALS shows a high degree of primary sequence homology with pyruvate carboxylase and pyruvate oxidase the ubiquinone cofactors of pyruvate oxidase inhibit ALS, and it has been proposed that the ubiquinone-binding site of the ancestral enzyme also is the site of both SMM and imidazolinone binding. Recent evidence suggests, however, that these two types of herbicide interact differently with ALS (a) imidazolinones cause a rapid decrease in the levels of extractable ALS activity in maize, whereas SMM does not and can protect the ALS activity from this in vivo effect of imidazolinones and (b) not all imidazolinone-tolerant cell lines are insensitive to sulfonylureas. Sulfonylureas and imidazolinones do not, however, show synergistic inhibition of maize ALS in vitro. ... 

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