Abstract / Introduction:
The
metabolism of glucose begins in the cytoplasm with glycolysis, a
pathway that converts
the monosaccharide into 2 molecules of pyruvate. Pyruvate is then
converted into acetyl-CoA which enters the TCA (tricarboxylic
acid) cycle in the mitochondria. This cycle, also known
as the citric acid cycle or Krebs
Cycle, was named for Hans Krebs, one of the scientists involved in its
elucidation in the 1930's (1). The cycle oxidizes pyruvate to CO2,
reducing NAD+ and FAD to NADH and FADH2,
respectively. These coenzymes are involved in the production of
ATP via
the electron transport chain and oxidative phosphorylation. The link
between Glycolysis and the TCA cycle involves the conversion of
pyruvate to acetyl-CoA. This reaction is catalyzed by a multi-enzyme
complex called the pyruvate dehydrogenase complex, a sixty
subunit complex involving three enzymes: 24 pyruvate dehydrogenase
(E1), 24
dihydrolipoyl transacetylase (E2), and 12 dihydrolipoyl dehydrogenase
(E3); the enzymes are associated noncovalently (1). The complex also
contains two loosely associated regulatory proteins: PDH kinase and PDH
phosphatase. The complex is
tightly regulated, both by its products acetyl-CoA and NADH, as well as
reversible phosphorylation by the associated kinase and phosphatase.
The
latter two enzymes specifically act on the pyruvate
dehydrogenase enzyme.
The
reactions catalyzed by the complex involve multiple coenzymes: thiamine
pryophosphate, coenzyme A, lipoic acid, NAD+ and FAD. The
overall reaction is (1):
Pyruvate + CoA + NAD+ → acetyl-CoA + CO2 + NADH + H+
Pyruvate dehydrogenase (E1) is a
heterotetramer (alpha2 beta2) with a total weight of 154
kDa (2). The enzyme has two catalytic sites and two cofactors,
magnesium ion and thiamine pyrophosphate (2). The
pyruvate dehydrogenase enzyme (E1) catalyzes the first step in the
reaction (3):
pyruvate
→ pyruvate-lipoamide-E2
This
reaction is actually a two-step process involving the binding of
pyruvate to thiamine
pyrophosphate (TPP) and the subsequent decarboxylation of pyruvate,
leading to a resonance stabilized carbanion intermediate that is
hydrated to form hdyroxyethyl-TPP. Hydroxyetheyl-TPP reacts with lipoic
acid, which is bound to the second enzyme, dihydrolipoyl
transacetylase. Dihydrolipoyl transacetylase and dihydrolipoyl
dehydrogenase then continue the reaction to completion to form
acetyl-CoA (2). The role of the individual subunits (alpha and beta) of
the pyruvate
dehydrogenase enzyme is still speculative, but it is proposed that both
participate in the binding of thiamine pyrophosphate (3).
References:
1. Garret, R.H. and Grisham, C.M. (1999) Biochemistry, 2nd Ed.
Brooks/Cole-Thomson Learning, Inc. Pacific Grove, CA
2. Ciszak, E.M. et al. (2003) J. Biol. Chem. 278, 21240-21246
3. Korotchkina, L.G., Ali, M.S. and Patel, M.S. (1996) Probing the
active site of mammalian pyruvate dehydrogenase. in Alpha-keto acid
dehyrogenase complexes (M. Patel, T. Roche, R. Harris eds.) Birkhauser
Verlag, pp 17-32.