Dehydrogenases half-site reactivity

Dehydrogenases carboxyraethylation, 1015 cobalt substituted, 1012 coenzymes binding, 1010 denaturation, 1016 half-site reactivity, 1011 inhibitors binding, 1016... 

Zhou J, Weiner H. Basis for half-of-the-sites reactivity and the dominance of the K487 oriental subunit over the E487 subunit in heterotetrameric human liver mitochondrial aldehyde dehydrogenase. Biochemistry 2000 39 12019-12024... 

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 Our own kinetic NMR studies on human PDH El (unpublished) support the model suggested, but similar studies on related thiamin enzymes, such as pyruvate decarboxylase, transketolase or pyruvate oxidase reveal that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). 

The best-studied example of a CoA-dependent nonphosphorylating ALDH is the methylmalonate-semi-aldehyde dehydrogenase, which has been isolated from both mammalian and bacterial sources. This enzyme transforms malonate semialdehyde and methylmalonate semialdehyde into acetyl-CoA and propionyl-CoA, respectively, through an oxidation reaction as described above, followed by a decarboxylation reminiscent of other (3-keto acids. Mechanistic studies of the B. sukilis enzyme have shown that it is activated by NAD" " binding, that it exhibits half-of-sites reactivity (only two moles of NADH forms per tetrameric protein unit) and that the decarboxylation reaction occurs after formation of the acyl-enzyme intermediate. Acyl transfer from the enzyme to CoA completes the reaction. 

Seifert F, Golblk R, Brauer J et al (2006) Direct kinetic evidence for half-of-the-sites reactivity in the El component of the human pyruvate dehydrogenase multienzyme complex through alternating sites cofactor activation. Biochemistry 45 12775-12785... 

The reductive half-reaction of methylamine dehydrogenase is shown in Scheme 10. The methylamine substrate initiates a nucleophilic attack on the quinone carbon at the C6 position of the TTQ cofactor displacing the oxygen to form a substrate-TTQ Schiff base adduct (29). The reactivity of the C6 position was demonstrated by covalent adduct formation at this position by hydrazines which are inactivators of methylamine dehydrogenase. Deprotonation of the substrate-derived carbon of 29 by an active-site amino acid residue results in reduction of the cofactor and yields an intermediate in which the Schiff base is now between the nitrogen and substrate-derived carbon (30). Hydrolysis of 30 releases the formaldehyde product and yields the aminoquinol form of the cofactor with the substrate-derived amino group still covalently bound (31). 

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