Cofactors & Vitamins
Andy Howard
Introductory Biochemistry
11 November 2008
Biochem: Cofactors,Vitamins
11/11/2008
What we’ll discuss

Coenzymes







NAD and NADP
FAD and FMN
Coenzyme A
TPP, PLP
Biotin, THF
Cobalamin
Lipoamide
Biochem: Cofactors,Vitamins

Vitamins


Why they exist
Water-soluble
vitamins



Cofactor precursors
Ascorbate
Fat-Soluble
Vitamins
11/11/2008
Page 2 of 49
Major cosubstrates (review)


Facilitate group transfers, mostly small groups
Oxidation-reduction participants
Cosubstrate
ATP
S-adenosylMet
UDP-glucose
NAD,NADP
Coenzyme A
Tetrahydrofolate
Ubiquinone
Source
Function
Transfer P,Nucleotide
Methyl transfer
Glycosyl transfer
Niacin
2-electron redox
Pantothenate Acyl transfer
Folate
1Carbon transfer
Lipid-soluble e- carrier
Biochem: Cofactors,Vitamins
11/11/2008
Page 3 of 49
Major prosthetic groups (rvw)


Transfer of larger groups
One- or two-electron redox changes
Prosth.gp.
FMN, FAD
TPP
PLP
Biotin
Adenosylcobalamin
MeCobal.
Lipoamide
Retinal
Vitamin K
Source
Riboflavin
Thiamine
Pyridoxine
Biotin
Cobalamin
Function
1e- and 2e- redox transfers
2-Carbon transfers with C=O
Amino acid group transfers
Carboxylation, COO- transfer
Intramolec. rearrangements
Cobalamin
Methyl-group transfers
Transfer from TPP
Vision
Carboxylation of glu residues
Vitamin A
Vitamin K
Biochem: Cofactors,Vitamins
11/11/2008
Page 4 of 49
NAD+ and NADP+


Net charge isn’t really >0 ;
the + is just a reminder that the
nicotinamide ring is positively charged
Most important cosubstrates in oxidationreduction reactions in aerobic organisms
Structure courtesy of
Sergio Marchesini, U.
Brescia
Biochem: Cofactors,Vitamins
11/11/2008
Page 5 of 49
Differences between them



The chemical difference is in the
phosphorylation of the 2’ phosphate
group of the ribose moiety
The functional difference is that NAD+
is usually associated with catabolic
reactions and NADP+ is usually
associated with anabolic reactions
Therefore often NAD+ and NADPH are
reactants and NADH and NADP+ are
products
Biochem: Cofactors,Vitamins
11/11/2008
Page 6 of 49
How do we get back to the
starting point?


NADH is often oxidized back to NAD+ as
part of the electron-transport chain
Imbalances can be addressed via:


NAD Kinase (S.Kawai et al (2005),
J.Biol.Chem. 280:39200)
NADP phosphatase
Biochem: Cofactors,Vitamins
11/11/2008
Page 7 of 49
iClicker quiz: question 1





What would you expect to be the
phosphate donor in the NAD kinase
reaction?
(a) free phosphate
(b) pyrophosphate
(c) ATP
(d) pyridoxal phosphate
Biochem: Cofactors,Vitamins
11/11/2008
Page 8 of 49
Reduced forms of NAD(P)



Reduction occurs on the
nicotinamide ring
Ring is no longer netpositive
Ring is still planar but
the two hydrogens on
the para carbon are not
Biochem: Cofactors,Vitamins
11/11/2008
Page 9 of 49
FAD and FMN






Flavin group based on riboflavin
Alternate participants in redox reactions
Prosthetic groups: tightly but noncovalently
bound to their enzymes
That protects against wasteful reoxidation of
reduced forms
FADH2 is weaker reducing agent than NADH
These are capable of one-electron oxidations
and reductions
Biochem: Cofactors,Vitamins
11/11/2008
Page 10 of 49
FAD and FMN structures

FAD has an AMP attached P to P
Structure courtesy
Paisley University
Biochem: Cofactors,Vitamins
11/11/2008
Page 11 of 49
FMN/FAD redox forms

Two-electron version: H+ + :H- transferred
Reaction diagram courtesy of Eric
Neeno-Eckwall, Hamline University
Biochem: Cofactors,Vitamins
11/11/2008
Page 12 of 49
(ADP-3’P)
Coenzyme A



Reactive portion
is free sulfhydryl
at one end of
the molecule
Can form
thioester with
acetate, etc.
Pantoate +
b-alanine =
pantothenate
Biochem: Cofactors,Vitamins
(Pantoate)
2-mercaptoethylamine)
b-alanine)
Structure courtesy of
MPB project, George
Washington University
11/11/2008
Page 13 of 49
Thiamine Pyrophosphate




Based on thiamine, vitamin B1
Many carboxylases and oxidative
decarboxylases use this coenzyme
So do transketolases (move 2 carbons
at a time between sugars with keto
groups)
Thiazolium ring is reactive center:
pKa drops from 15 in H2O to 6 in
enzyme
Biochem: Cofactors,Vitamins
11/11/2008
Page 14 of 49
TPP reactions
pyrimidine
thiazolium
Diagram courtesy of
Oklahoma State U.
Biochemistry program
Biochem: Cofactors,Vitamins
11/11/2008
Page 15 of 49
Pyridoxal
phosphate



PLP is prosthetic group for many
amino-acid-related enzymes,
particularly transaminations
Carbonyl group of PLP bound as a
Schiff base (imine) to -amino
group of lysine at active site
First step is always formation of
external aldimine; goes through
gem-diamine intermediate to
internal aldimine
Biochem: Cofactors,Vitamins
11/11/2008
Page 16 of 49
Transaminations


-amino acid1 +
-ketoacid2 
-ketoacid1 +
-amino acid2
Example:





-amino acid1 = glutamate
-ketoacid2 = pyruvate
-ketoacid1 = a-ketoglutarate
-amino acid2 = alanine
Many biosynthetic / degradative pathways for
normal amino acids depend on these reactions
Biochem: Cofactors,Vitamins
11/11/2008
Page 17 of 49
Biotin



Rarity: vitamin is the prosthetic group
Used in reactions that transfer carboxyl
groups
… and in ATP-dependent carboxylations
Biochem: Cofactors,Vitamins
11/11/2008
Page 18 of 49
Biotin reactivity


Covalently bound to active-site lysines to
form species called biocytin
Pyruvate carboxylase is characteristic
reaction:
Diagram courtesy
University of Virginia Biochemistry
Biochem: Cofactors,Vitamins
11/11/2008
Page 19 of 49
Tetrahydrofolate



Primary donor of one-carbon units
(formyl, methylene, methyl)
Supplies methyl group for thymidylate
Dihydrofolate reductase (DHFR) is an
interesting drug target


Methotrexate as cancer chemotherapeutic:
cancer needs more thymidylate than healthy cells
Trimethoprim as antibacterial:
Bacterial DHFR is somewhat different from
eucaryotic DHFR because bacteria derive DHF
from other sources; humans get it from folate
Biochem: Cofactors,Vitamins
11/11/2008
Page 20 of 49
THF structure and function
Figure courtesy
horticulture program,
Purdue
Biochem: Cofactors,Vitamins
11/11/2008
Page 21 of 49
Cobalamin




Largest B vitamin
Corrin ring structure related to heme but
missing one carbon in ring structure
Cobalt bound in core of ring system
Involved in enzymatic rearrangements



Catabolism of odd-chain fatty acids
Methylation of homocysteine
Reductive dehalogenation
Biochem: Cofactors,Vitamins
11/11/2008
Page 22 of 49
AdenosylCobalamin
Reactive
Co-C bond
“Missing” carbon
Diagram courtesy of
Swiss Food News
Biochem: Cofactors,Vitamins
11/11/2008
Page 23 of 49
Lipoamide






Protein-bound form of lipoic acid
Contains five-membered disulfide ring
Covalently bound via amide to protein
lysine sidechain
Involved in swinging arm between active
sites in multienzyme complexes
Disulfides break periodically
Example: pyruvate dehydrogenase
complex
Biochem: Cofactors,Vitamins
11/11/2008
Page 24 of 49
Lipoamide 2e- reduction

Cf. Scheme 7.6: thioester starting point
Fig. Courtesy Biochem
and Biophysics
program, Rensselaer
Biochem: Cofactors,Vitamins
11/11/2008
Page 25 of 49
iClicker revisited

Which coenzyme would you expect
would be required for the reaction
oxaloacetate + glutamate 
aspartate + -ketoglutarate?
(a) ascorbate
(b) PLP
( c) thiamine pyrophosphate
(d) NAD
(e) none of the above
Biochem: Cofactors,Vitamins
11/11/2008
Page 26 of 49
Many cofactors are
derived from vitamins

We justify lumping these two topics
together because many cofactors are
vitamins or are metabolites of vitamins.
Biochem: Cofactors,Vitamins
11/11/2008
Page 27 of 49
Vitamins: necessary
micronutrients that cannot be
synthesized internally



What’s a vitamin for one organism is not
for another
Primates and some rodents are the only
vertebrates that don’t synthesize
ascorbate
E.coli can make almost everything given
energy and sources of atoms
Biochem: Cofactors,Vitamins
11/11/2008
Page 28 of 49
Why wouldn’t organisms
make everything?



Complex metabolites require energy for
synthesis
Control of their synthesis is also
metabolically expensive
Cheaper in the long run to derive these
nutrients from diet
Biochem: Cofactors,Vitamins
11/11/2008
Page 29 of 49
Vitamins: broad classifications

Water-soluble vitamins



Coenzymes or coenzyme precursors
Non-coenzymic metabolites
Fat-soluble vitamins


Antioxidants
Other lipidic vitamins
Biochem: Cofactors,Vitamins
11/11/2008
Page 30 of 49
Are all nutrients that we can’t
synthesize considered
vitamins?



No:
If it’s required in large quantities,
it’s not a vitamin
By convention, essential fatty acids like
arachidonate aren’t considered vitamins
Biochem: Cofactors,Vitamins
11/11/2008
Page 31 of 49
Coenzyme precursors


We’ve already outlined the fact that most
water-soluble coenzymes are derived
from vitamins—typically B vitamins
Typically the dietary form can be
converted by a fairly short metabolic
pathway into the coenzyme form, e.g.


niacin + glutamine 
nicotinamide + glutamate
nicotinamide + ADP-ribose  NAD
Biochem: Cofactors,Vitamins
11/11/2008
Page 32 of 49
The B vitamins









All aqueous micronutrients
Generally identified via pathologies associated
with dietary deficiencies
B1: thiamin (produces TPP)
B2: riboflavin (produces FAD, FMN)
B3: niacin (produces NAD, NADP)
B5: pantothenate (produces Coenzyme A)
B6: pyridoxamine (produces PLP)
B9: folate: produces THF, THF derivatives
B12: cobalamin (produces adenosylcobalamin,
methylcobalamin)
Biochem: Cofactors,Vitamins
11/11/2008
Page 33 of 49
Deficiency of niacin



Pellagra: dermatitis, diarrhea,
dementia
Still found in some diets that are low
in vitamin content
Humans can actually synthesize
nicotinamide from tryptophan; but
that’s often in short supply too
Biochem: Cofactors,Vitamins
11/11/2008
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Image courtesy
history.nih.gov
Page 34 of 49
Deficiency of thiamine


Beriberi: primary symptoms
are in nervous system and
musculature
Polished rice is missing
thiamine;
rice hulls are rich in it
Biochem: Cofactors,Vitamins
11/11/2008
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Image courtesy
answers.com
Page 35 of 49
Deficiency of cobalamin




Available sources of cobalamin are animal
products
Therefore vegans need to be careful to get
cobalamin from supplements
Symptoms of deficiency (pernicious anemia):
weakness, fatigue, pallor, palpitations, dizziness
Deficiency is common even in non-vegans:
5-40% of the population?
Biochem: Cofactors,Vitamins
11/11/2008
Page 36 of 49
Ascorbate





Vitamin in primates, some rodents
Synthesizable in most other vertebrates
Involved in collagen processing
Reduced form acts as reducing agent
during hydroxylation of collagen
Deficiency gives rise to inadequate
collagen - scurvy
Biochem: Cofactors,Vitamins
11/11/2008
Page 37 of 49
PTM role of ascorbate
(fig. 6.17)

Proline + O2 + -ketoglutarate + ascorbate 
4-hydroxyproline + succinate + CO2 +
dehydroascorbate

This is a post-translational modification that
occurs to prolines within collagen
The hydroxylated prolines help stabilize the
collagen triple helix
Hydroxylysine found in collagen too


Biochem: Cofactors,Vitamins
11/11/2008
Page 38 of 49
Dietary deficiency
of ascorbate


Primary sources of
ascorbate are fruits,
particularly citrus, and
green vegetables
Ascorbate deficiency’s
first symptom involves
collagen degradation,
leading to scurvy
Biochem: Cofactors,Vitamins
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Image courtesy
U.Cincinnati
Medical School
11/11/2008
Page 39 of 49
Scurvy in history


Shortage of green vegetables in
sailors’ diets meant scurvy was
rampant on shipboard until the
18th century
Success of English navy over
French 1760-1800 was partly due
to the introduction of limes in
English sailors’ diets 50 years
before the French caught on
Biochem: Cofactors,Vitamins
11/11/2008
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Page 40 of 49
Lipid vitamins





Contain rings & long aliphatic
sidechains
At least one polar group in
each
Absorbed in intestine, carried
via bile salts
Hard to study
Most are formally built from
isoprene units, as are steroids
Biochem: Cofactors,Vitamins
11/11/2008
Page 41 of 49
Vitamin A (retinol)



3 forms varying in terminal polar group
Involved in signaling and receptors
b-carotene is nonpolar dimer
Biochem: Cofactors,Vitamins
11/11/2008
Page 42 of 49
Vitamin A deficiency


Produces night blindness because the
retina and cornea dry out
Most common cause: nursing infants
whose mothers have vitamin A deficiency
in their diet
Biochem: Cofactors,Vitamins
11/11/2008
Page 43 of 49
Vitamin D


Several related forms
Hormones involved in
Ca2+ regulation
Figure courtesy
Cyberlipid
Biochem: Cofactors,Vitamins
(cholecalciferol)
11/11/2008
Page 44 of 49
Vitamin D deficiency



Rickets in children:
Bone disease, restlessness, slow growth
One form of vitamin D is actually
synthesizable from cholesterol given
adequate sunlight;
Therefore rickets is most common in
densely settled urban environments
Biochem: Cofactors,Vitamins
11/11/2008
Page 45 of 49