Coenzymes
• Many enzymes contain small nonprotein
molecules and metal ions
• Coenzyme
– a complex organic or metalloorganic molecule
• Prosthetic groups
– are distinguished by their tight, stable incorporation
into a protein’s structure by covalent or noncovalent
forces.
– Examples
• Pyridoxal phosphate,flavin mononucleotide (FMN), flavin
dinucleotide (FAD), thiamin pyrophosphate, biotin, and the
metal ions.
• Some enzymes require both a coenzyme and
one or more metal ions for activity
• Coenzymes
– participate directly in substrate binding or
catalysis.
• A complete, catalytically active enzyme
together with its bound coenzyme and/or
metal ions is called a holoenzyme. The protein
part of such an enzyme is called the
apoenzyme or apoprotein.
• Intracellular range of concentration
– 0.01 -1.0 meq L-1
• Coenzymes may be covalently bound to the
enzyme or free to associate and dissociate
from the protein.
• may facilitate the binding and orientation of
substrates
• the formation of covalent bonds with reaction
intermediates (Co2+ in coenzyme B12)
• interaction with substrates to render them
more electrophilic (electron-poor) or
nucleophilic (electron-rich).
• Coenzymes serve as recyclable shuttles—or group
transfer reagents—that transport many substrates
from their point of generation to their point of
utilization.
– Other chemical moieties transported by coenzymes
include methyl groups (folates), acyl groups (coenzyme A),
and oligosaccharides (dolichol).
• Association with the coenzyme also stabilizes
substrates such as hydrogen atoms or hydride ions that
are unstable in the aqueous environment of the cell.
• Chemical reactions of many types take place
between substrates and enzymes’ functional
groups (specific amino acid side chains, metal
ions, and coenzymes).
• Catalytic functional groups on an enzyme may
form a transient covalent bond with a
substrate and activate it for reaction, or a
group may be transiently transferred from the
substrate to the enzyme.
• A number of amino acid side chains, and the
functional groups of some enzyme cofactors
can serve as nucleophiles in the formation of
covalent bonds with substrates.
• The covalent bond formed can activate a
substrate for further reaction
Coenzyme role
• Epimerization involves an oxidation and
reduction at carbon 4 with NAD+ as coenzyme.
• Each step in fatty acid oxidation involves acylCoA derivatives catalyzed by separate
enzymes, utilizes NAD+ and FAD as coenzymes,
and generates ATP
• Susceptibility to proteolytic degradation can
be influenced by the presence of ligands such
as substrates, coenzymes, or metal ions that
alter protein conformation
• NAD is also required for the poly-ADP-ribose
polymerase reaction, which is part of the
cellular DNA damage recognition system and
regulates DNA replication, DNA repair, and cell
cycle progression.
• Metabolic pathways are regulated at several
levels, from within the cell and from outside
– The most immediate regulation is by the
availability of substrate
– A second type of rapid control from within is
allosteric regulation by a metabolic intermediate
or coenzyme—an amino acid or ATP, for
example—that signals the cell’s internal metabolic
state.
• Many coenzymes, cofactors, & prosthetic
groups are derivatives of B vitamins
• Many coenzymes contain, in addition, the
adenine, ribose, and phosphoryl moieties of
AMP or ADP.
Many coenzymes and related compounds are derivatives of
adenosine monophosphate.
Synthesis
• They are synthesized by a variery of ammalian
cell types
• Synthesis of nucleotide coenzymes is
regulated so that there are essentially
constant concentrations of these coenzymes
in the cell.
• Synthesis of niacin requires pyridoxine,
riboflavin, and iron.
• The function of coenzymes is chemically
varied, and we describe each separately
• The coenzyme pyridoxal phosphate (PLP) is
present at the catalytic site of
aminotransferases and of many other
enzymes that act on amino acids
• In each case, the thiamin diphosphate provides a
reactive carbon on the thiazole moiety that forms
a carbanion, which then adds to the carbonyl
group of, for instance, pyruvate. The addition
compound then decarboxylates, eliminating CO2.
• Electrical stimulation of nerve leads to a fall in
membrane thiamin triphosphate and release of
free thiamin. It is likely that thiamin triphosphate
acts as a phosphate donor for phosphorylation of
the nerve membrane sodium transport channel.
• NAD Is the Source of ADP-Ribose
– ADP-ribosylation
– DNA repair mechanism
• Vitamin B6 Has Several Roles in Metabolism
– transamination and decarboxylation
– glycogen phosphorylase, where the phosphate group
is catalytically important
– in steroid hormone action
• where it removes the hormone-receptor complex from DNA
binding, terminating the action of the hormones.
• A few types of coenzymes and proteins serve
as universal electron carriers
• Both NAD and NADP accept two electrons and
one proton.
• the coenzymes function catalytically and are
recycled repeatedly without a net change in
the concentration of NAD NADH.
• NAD and NADP are the freely diffusible
coenzymes of many dehydrogenases.
• NAD or NADP accepts a hydride ion (:H-, the equivalent of a proton and
two electrons)
• the coenzymes function catalytically and are recycled repeatedly without a
net change in the concentration of NAD+ + NADH.
• NAD and NADP are bound to dehydrogenases
in a widely conserved structural motif called
the Rossmann fold.
• Coenzymes play key roles In the citric acid
cycle
– energy-yielding metabolism
• NAD(P)+-dependent dehydrogenases are
assayed spectrophotometrically
– the rate of change in optical density at 340 nm will
be proportionate to the quantity of enzyme
present
• Many enzymes that do not directly reduce
NAD+ or FAD generate products that can be
acted upon by a NAD(P) or FAD-linked
dehydrogenase. Thus by coupling two enzyme
reactions
Sirtuins
• The sirtuins are a highly conserved family of
NAD+-dependent enzymes
• sirtuins serve as the bridge between what we
eat and what we are.
• Sirtuin activity is intimately tied to the
metabolic state of the cell.
– Linking Chromatin Remodeling to Metabolism
• requires NAD+ cleavage with each reaction
cycle
• flavoproteins as electron carriers
• Certain flavoproteins act in a quite different
role as light receptors.
– Mediate the effects of light on mammalian
circadian rhythms (oscillations in physiology and
biochemistry, with a 24-hour period)
• Photolyases use the energy of absorbed light to repair
chemical defects in DNA.
• Vitamin C Is the Coenzyme for Two Groups of
Hydroxylases
– Copper-containing hydroxylases
– α-ketoglutarate-linked iron-containing
hydroxylases
CLINICAL CORRELATION
• Cystathioninuria
• Mutation of a coenzyme-binding site results in
clinical disease
– y-cystathionase
• Cystathionine → cysteine + α-ketobutyrate
• the Km for pyridoxal phosphate binding to the
enzyme was increased
Arsenic Poisoning
• For the most part, arsenic poisoning is
explained by inhibition of enzymes that
require lipoic acid as a coenzyme. These
include pyruvate dehydrogenase, αketoglutarate dehydrogenase, and branchedchain a-keto acid dehydrogenase.
Deficiency of vitamines
• limited diets, when food is cooked at high
temperatures for long periods,
• intestinal diseases, Inability to absorb
• in newborns , pregnant women
• Folic Acid Deficiency
– Altered appearance of blood cells and
formiminoglutamate excretion
• methyl malonic aciduria
– Acidosis
– 5’-adenosylcobalamin deficiency (coenzyme of
methyl malonyl CoA isomerization).
• methylmalonic aciduria
Assay of vitamines(coenzyme)
• by measuring one or more enzyme activities in
isolated red blood cells
As a tool for enzyme Purification
• Stationary phase matrices available
commercially contain ligands such as NAD+ or
ATP analogs.
• Bound proteins are then eluted either by
competition with soluble ligand or, less
selectively, by disrupting protein-ligand
interactions using urea, guanidine
hydrochloride, mildly acidic pH, or high salt
concentrations.