Cellular respiration occurs in three major stages (Fig. 15-1). In
the first stage, organic fuel molecules-glucose, fatty acids, and some amino
acids-are oxidized to yield two-carbon fragments in the form of the acetyl group
of acetyl-coenzyme A (acetyl-CoA). In the second stage, these acetyl groups are
fed into the citric acid cycle, which enzymatically oxidizes them to
CO2. The energy released by oxidation is conserved in
the reduced electron carriers NADH and FADH2. In the third stage of respiration,
these reduced cofactors are themselves oxidized, giving up protons
(H+) and electrons. The electrons are transferred
along a chain of electron-carrying molecules, known as the respiratory chain, to
O2, which they reduce to form
H2O. During this process of electron transfer much
energy is released and conserved in the form of ATP, in the process called
oxidative phosphorylation. Respiration is more complex than glycolysis and is
believed to have evolved much later, after the appearance of cyanobacteria,
which added oxygen-the electron acceptor of respiration-to the earth's
atmosphere.
We will discuss the third stage of cellular respiration (electron transfer
and oxidative phosphorylation) in Chapter 18. In this chapter we examine the
complete oxidation of pyruvate and the citric acid cycle, also
called the tricarboxylic acid cycle or the Krebs
cycle (after its discoverer). We consider first the cycle reactions and
the enzymes that catalyze them. Because intermediates of the citric acid cycle
are often used as biosynthetic precursors, some means of replenishing these
intermediates is essential to the continued operation of the cycle; we discuss
several such replenishing reactions. The mechanisms that regulate the flux of
material through the citric acid cycle are then considered. Finally, we describe
a metabolic sequence, the glyoxylate pathway, that employs some of the same
enzymes and reactions that occur in the citric acid cycle to enable the net
synthesis of glucose from stored triacylglycerols.
In aerobic organisms, glucose and other sugars, fatty acids, and most of the
amino acids are ultimately oxidized to CO2 and
H2O via the citric acid cycle. Before they can enter
the cycle, the carbon skeletons of sugars and fatty acids must be degraded to
the acetyl group of acetyl-CoA, the form in which the citric acid cycle accepts
most of its fuel input. Many amino acid carbons also enter the cycle this way,
although several amino acids are degraded to other intermediates of the cycle.
In Chapters 16 and 17, respectively, we shall see how fatty acids and amino
acids enter the citric acid cycle. Here we consider how pyruvate, derived from
glucose by glycolysis, is oxidized to yield acetyl-CoA and
CO2 by a structured cluster of three enzymes, the
pyruvate dehydrogenase complex, located in the mitochondria of
eukaryotic cells and in the cytosol of prokaryotes.
A careful examination of this enzyme complex can be rewarding in several
respects. The pyruvate dehydrogenase complex is a classic example, very well
studied, of a multienzyme complex in which a series of chemical intermediates
remain bound to the surface of the enzyme molecules as the substrate is
transformed into the imal product. Five cofactors participate in this key
reaction mechanism, all of which are coenzymes derived from vitamins. The
regulation of this enzyme complex also illustrates how a combination of covalent
modification and allosteric regulation results in precisely regulated flux
through a metabolic step. Finally, the pyruvate dehydrogenase complex is the
prototype for two other important enzyme complexes that we will encounter:
α-ketoglutarate dehydrogenase (of the citric acid cycle) and the branched-chain
α-ketoacid dehydrogenase involved in the oxidative degradation of several amino
acids (Chapter 17). The remarkable similarity in the protein structure, cofactor
requirements, and reaction mechanisms of these three complexes doubtless
reflects a common evolutionary origin.
Pyruvate Is Oxidized to Acetyl-CoA and CO2
The overall reaction catalyzed by the pyruvate dehydrogenase
complex is oxidative decarboxylation, an irreversible oxidation
process in which the carboxyl group is removed from pyruvate as a molecule of
CO2 and the two remaining carbons become the acetyl
group of acetyl-CoA (Fig. 15-2). The NADH formed in this reaction gives up a
hydride ion with its two electrons ( : H-) to the
respiratory chain (Fig. 15-1), which carries the electrons to oxygen or, in
anaerobic microorganisms, to an alternative electron acceptor such as sulfur.
This electron transfer to oxygen ultimately generates three molecules of ATP per
pair of electrons.
The irreversibility of the pyruvate dehydrogenase reaction has been proved by
isotopic labeling experiments: radioactively labeled
CO2 cannot be reattached to acetyl-CoA to yield
pyruvate labeled in the carboxyl group.
The combined dehydrogenation and decarboxylation of pyruvate to acetyl-CoA
(Fig. 15-2) involves the sequential action of three different enzymes, as well
as five different coenzymes or prosthetic groupsthiamine pyrophosphate (TPP),
flavin adenine dinucleotide (FAD), coenzyme A (CoA), nicotinamide adenine
dinucleotide tNAD), and lipoate. Four different vitamins required in human
nutrition are vital components of this system: thiamin (in TPP), riboflavin (in
FAD), niacin (in NAD), and pantothenate (in coenzyme A).
We have already described the roles of FAD and NAD as electron carriers
(Chapter 13), and we have encountered TPP as the coenzyme of the enzyme that
catalyzes decarboxylation of pyruvate to acetaldehyde in alcohol fermentation
(see Fig. 14-9). Pantothenate, present in all living organisms, is an essential
component of coenzyme A (Fig. 15-3). Coenzyme A has a reactive thiol (-SH) group
that is critical to its role as an acyl carrier in a number of metabolic
reactions; acyl groups become covalently linked to this thiol group, forming
thioesters. Because of their relatively high free energy of
hydrolysis (see Figs. 13-6, 13-7), thioesters have a high acyl group transfer
potential, donating their acyl groups to a variety of acceptor molecules. The
acyl group attached to coenzyme A may thus be thought of as activated for group
transfer.
The fifth cofactor for the pyruvate dehydrogenase reaction,
Iipoate (Fig. 15-4), has two thiol groups, both essential to
its role as cofactor. In the reduced form of lipoate both sulfur atoms are
present as -SH groups, but oxidation produces a disulfide (-S-S-) bond, similar
to that between two Cys residues in a protein. Because of this capacity to
undergo oxidation-reduction reactions, lipoate can serve both as an electron
carrier and as an acyl carrier; both functions are important in the action of
the pyruvate dehydrogenase complex.
The Pyruvate Dehydrogenase Complex Consists of Three Distinct Enzymes
The pyruvate dehydrogenase complex consists of multiple copies of each of the
three enzymes pyruvate dehydrogenase
(E1), dihydrolipoyl transacetylase
(E2), and dihydrolipoyl dehydrogenase
(E3) (Table 15-1). The number of copies of each
subunit, and therefore the size of the complex, varies from one organism to
another. The pyruvate dehydrogenase complex isolated from E. coli
(Mr > 4.5 × 106) is about 45 nm in
diameter, slightly larger than a ribosome, and can be visualized with the
electron microscope (Fig. 15-5a). The "core" of the cluster, to which the other
enzymes are attached, is dihydrolipoyl transacetylase
(E2). In the complex from E. coli, 24 copies
of this polypeptide chain, each containing three molecules of covalently bound
lipoate, constitute this core. In the complex from mammals, there are 60 copies
of E2 and six of a related protein, protein X, which
also contains covalently bound lipoate. The attachment of lipoate to the ends of
Lys side chains in E2 produces lipoyllysyl groups
(Fig. 15-4)-long, flexible arms that can carry acetyl groups from one active
site to another in the pyruvate dehydrogenase complex. Bound to the core of
E2 molecules are 12 copies of pyruvate dehydrogenase
(E1), each composed of two identical subunits, and six
copies of dihydrolipoyl dehydrogenase (E3), each also
composed of two identical subunits. Pyruvate dehydrogenase
(E1) contains bound TPP, and dihydrolipoyl
dehydrogenase (E3) contains bound FAD. Tvvo regulatory
proteins that are also part of the pyruvate dehydrogenase complex (a protein
kinase and a phosphoprotein phosphatase) will be discussed below.
Figure 15-6 shows schematically how the pyruvate dehydrogenase
complex carries out the five consecutive reactions in the decarboxylation and
dehydrogenation of pyruvate. Step 1 is essentially identical to the reaction
catalyzed by pyruvate decarboxylase (see Fig. 14-9c); C-1 of pyruvate is
released as CO2, and C-2, which in pyruvate has the
oxidation state of an aldehyde, is attached to TPP as a hydroxyethyl group. In
step 2 this group is oxidized to a carboxylic acid (acetate).
The two electrons removed in the oxidation reaction reduce the -S-S- of a
lipoyl group on E2 to two thiol (-SH) groups. The
acetate produced in this oxidation-reduction reaction is first esterified to one
of the lipoyl -SH groups, then transesterified to CoA to form acetylCoA (step
3). Thus the energy of oxidation drives the formation of a high-energy thioester
of acetate. The remainder of the reactions catalyzed by the pyruvate
dehydrogenase complex (steps 4 and 5) are electron transfers necessary to
regenerate the disulfide form of the lipoyl group of
E2 to prepare the enzyme complex for another round of
oxidation. The electrons removed from the hydroxyethyl group derived from
pyruvate eventually appear in NADH after passing through FAD.
Central to the process are the swinging lipoyllysyl arms of
E2, which pass the two electrons and the acetyl group
derived from pyruvate from El to
E3. All these enzymes and coenzymes are clustered,
allowing the intermediates to react quickly without ever diffusing away from the
surface of the enzyme complex. The five-reaction sequence shown in Figure 15-6
is another example of substrate channeling, as described
As one might predict, mutations in the genes for pyruvate dehydrogenase
subunits, or thiamin deficiency in the diet, have severe consequences.
Thiamin-deficient animals are unable to oxidize pyruvate normally, which is of
particular importance in the brain; this organ usually obtains all its energy
from the aerobic oxidation of glucose, and pyruvate oxidation is therefore
vital. Beriberi, a disease that results from dietary deficiency of thiamin, is
characterized by loss of neural function. This disease occurs primarily in
populations that depend on white (polished) rice for most of their food; white
rice lacks the hulls in which most of the thiamin of rice is found. People who
habitually consume large amounts of alcohol can also develop thiamin deficiency;
much of their dietary intake is in the form of the "empty (vitamin-free)
calories" of distilled spirits. An elevated level of pyruvate in the blood is
often an indicator of defects in pyruvate oxidation due to one of these causes
. |
|
|
