Download Nucleotides: Be able to differentiate between a purine ring and a

Nucleotides:
Be able to differentiate between a purine ring and a pyrimidine ring. Be able to name the two purine
nitrogenous bases and the three pyrimidine bases. Know the components of a nucleotide and a nucleoside.
Know the difference between a nucleotide and a deoxynucleotide? Note sugar numbering is with a ‘ (prime)
Brief Cofactor Review:
Do not get too hung up on the difference between cofactor and coenzyme. They are one author’s classification
and another author may use another. A cofactor is something that an enzyme needs in order to catalyze a
reaction. The enzyme can use the R groups of the amino acids in its chain, but sometimes something more is
sometimes needed. The cofactor is found at the active site of the enzyme and participates in the catalytic
mechanism.
A cofactor can be:
1) a metal
2) an organic molecule – we will call these coenzymes
1) The metal can be
A) an activator ion – these reversibly bind to the enzyme and are in equilibrium between the bound
and free form.
1) K+ present in high concentrations (135 mM) in the cell and there is little change in
concentration; proteins usually have a net (-) charge, K+ binds and stabilizes the
tertiary structure
2) Mg2+ required by kinases and is found bound to ATP; intracellular concentration is 1 mM
and doesn’t change a lot; is found to activate some enzymes
3) Ca2+ important signaling molecule; low concentrations in the cell (100 nM to 1 μM) and there
are significant changes in concentration; binds to a specific site on an enzyme and
activates it
B) an integral part of the enzyme – as in a metalloenzyme
These metals are tightly bound, participate in the catalytic mechanism, and stay with the
enzyme as a prosthetic group
1) Fe2+ participates in electron transfer; is present either in a heme group or as an iron-sulfur
complex
2) Zn2+ is involved in the catalytic mechanism and may stabilize the transition state; it may
abstract a proton from water forming the hydroxyl nucleophile
3) Cu2+ participates in electron transfer
4) Co2+, Ni2+, Mo2+, Mn2+ - minor amounts, participate in catalytic mechanisms
2)
A coenzyme is typically an organic molecule. Many are commonly known as the vitamins to you.
Coenzymes were divided into two groups; cosubstrates and prosthetic groups. Be able to identify if a coenzyme
is covalently or tightly linked to the enzyme. A cosubstrate in this definition is a coenzyme that leaves the
enzyme. In a reaction mechanism, we could also consider these as substrates. A prosthetic group is a
coenzyme that is “permanently” attached to the enzyme. It does not leave the enzyme.
Enzymes and cofactors are catalysts and do not get used up. They are regenerated. A cosubstrate can leave
the enzyme to be regenerated. A prosthetic group is regenerated while on the enzyme.
High energy molecules:
We will discuss this later, but for now, we will just call ATP a “high energy molecule”. In fact, any of the
nucleotides are high energy molecules, ATP is just the most common. I am using the term “activated” below
very loosely, this is in a biochemical sense, not a thermodynamic sense.
Note the 4 types of activation schemes employed by ATP. The result in each case is ultimately the formation of
a new bond.
1)
2)
3)
4)
It can be used to activate molecules by donating a phosphate. ATP is the predominant nucleotide that
does this. In the example provided to you, glucose-6-phosphate is formed. The enzyme doing this is a
kinase. Loosely, the energy present in ATP has been transferred to glucose-6-phosphate.
Pyrophospate donation is a second example
Nucleotides can be used to activate molecules by donating a nucleotide monophosphate group. In the
examples provided to you, UTP is used to activate glucose-1-phosphate and CTP is used to activate
phosphoethanolamine. CTP is typically used in lipid pathways and UTP is typically used in
carbohydrate pathways. The formation of 3’ Aminoacyl-tRNA is another example of this type. ATP is
used to link an amino acid to a tRNA. This enzyme is a ligase (t-RNA synthetase).
The last example is donation of the adenylyl group. This occurs in the formation of SAM.
As we progress though the coenzyme, you will note that ATP is involved in several ways, by either donating a
phosphate or an adenylyl group.
The rest of the story:
1) S-adenosylmethionine (SAM)
A)
B)
C)
D)
E)
a methyl group donor
dietary source is methionine
ATP is used as an adenosyl donor
the methyl of methionine is stable and not a target of nucleophilic attack, the methyl of SAM is
subject to nucleophilic attack and thus SAM can donate a methyl group.
The sulfur in SAM is postitively charged, a sulfonium ion.
After SAM has donated its methyl group, it becomes S-adenosylhomocysteine. The adenosyl
group is removed and it becomes homocysteine (like cysteine with an extra CH2). This is
converted back to SAM through the use of tetrahydrofolate (a coenzyme we cover later).
2) Phosphoadenosine Phosphosulfate
A)
B)
C)
D)
sulfate and phosphate in solution are stable and are not easily added to molecules.
sulfate is less stable when esterified to a phosphate and is activated by transfer of an AMP from
ATP to sulfate and release of pyrophosphate
in a manner analogous to the kinase reaction, where a phosphate from ATP is added to an OH
group, a sulfate from PAPS can be added to an OH (or amine) group. This occurs frequently
on the OH groups of sugars.
note that a second phosphate is added to the 3’ OH group of the ribose ring and after donation
of the sulfate, the final molecule is a bisphosphate, not a diphosphate
3) Nicotinamide adenine dinucleotide
A)
B)
C)
D)
E)
F)
G)
H)
I)
J)
sulfate group donor
involved in oxidation-reduction reactions
why is this called a dinucleotide? because there are two entire nucleotide moieties in the
molecule. One of the nitrogenous bases is adenine (so one of the nucleotides is adenosine
monophosphate) and the other nitrogenous base is nicotinamide.
a closely related molecule is nicotinamide adenine dinucleotide phosphate, which has a
phosphate group on the 2 OH of ribose.
Because one of the bases in these dinucleotides is nicotinamide and nicotinamide is a pyridine
base, these cofactors are often called the pyridine nucleotides.
Nicotinamide or a precursor, niacin, must be provided in the diet. Nicotinamide is the amide
form of niacin (known as vitamin B3).
In the oxidized form, NAD carries a positive charge. It is reduced to a neutral form by addition
of a hydride ion. As discussed before, this hydride is added stereospecifically to one face of the
nicotinamide ring. The reaction is NAD+ + reduced substrate ⇔ NADH + H+ + oxidized
substrate. Note that the equivalent of two hydrogens are removed from the substrate. One is
removed as a hydride and the other ends up as a free proton. Since a hydride is added to
NAD+, there is an obligatory 2 electron transfer.
The reduced form (NADH) absorbs light at 340 nm
NADH is relatively stable to air oxidation
NADH is loosely bound to the enzyme and leaves after the catalytic event is over.
NAD+ has a high reduction potential (- 0.32 ev)
lactate dehydrogenase was the enzyme example provided.
4) Flavin adenine dinucleotide
A)
B)
C)
D)
E)
F)
G)
H)
5)
B)
C)
D)
6)
involved in acyl and acetyl transfer
This coenzyme has three major portions:
a)
mercaptoethylamino group, which has the important sulfur
b)
pantothenic acid (which was originally called vitamin B5)
c)
ADP-3’ phosphate (note the involvement of something with the adenine
nitrogenous base)
There is formation of a thioester between the carboxylic acid function of a fatty acid and the
sulfur of coenzyme A.
Note that coenzyme A is hydrophilic and it is used to “solubilize” a hydrophobic fatty acid and
allows it to be transfer within the cell.
ATP is required to form the bond between the fatty acid and the sulfur group. The enzyme that
does this is a ligase.
Thiamine Pyrophosphate
A)
B)
C)
D)
7)
Riboflavin (vitamin B2) is a necessary dietary precursor. Riboflavin consists of a nitrogenous
base (isoalloxazine – don’t need to know this) and the sugar ribitol (like ribose but the aldehyde
is reduced to an alcohol)
this is also a dinucleotide, and one of the nucleotides has the adenine base. the other
nucleotide contains the nitrogenous base present in riboflavin.
A related molecule is flavin mononucleotide which is the phosphorylated riboflavin
The oxidation reduction occurs on the isoalloxazane ring. Both the oxidized and reduced forms
are neutral. Unlike NAD, there is a free radical intermediate and electron transfer can occur by
1 electron steps and can serve as an intermediary between other electron carriers that are
obligatory 2 (like NADH) or 1 (like Fe2+) electron carriers . The fully oxidized form is a quinone,
the form reduced by one electron is the semiquinone and the fully reduced form is the
hydroquinone. The fully reduced form has gained two hydrogen atoms (FADH2)
The oxidized form absorbs yellow light (450 nm)
FADH2 is not stable and is easily air oxidized.
FAD remains tightly bound to the enzyme and must be regerated on the enzyme
FAD has a smaller reduction potential than NAD+. The exact value is somewhat variable and
depends on the environment
Coenzyme A
A)
B)
C)
D)
involved in decarboxylations and transketolase/aldolase reactions
This is derived from the dietary precursor, thiamine (B1), which contains a pyrimidine ring and a
thiazolium ring. The thiazolium ring is positively charged with the charge residing on the
nitrogen.
This must be phosphorylated (by ATP) before it is functional.
The C-2 of the thiazolium ring has an altered pKa. It would normally be about 18, but in the
enzyme environment and near the (+) N, it can form the carbanion. The H is removed by a
base (often a histidine). The carbanion is a potent nucleophile. The compound with a – and a +
charge is referred to as an ylid.
The example given is pyruvate decarboxylase. Note the role of the enzyme conjugate base.
Note the fact that that the ring N can act to stabilize the carbanion. Note the fact that there is
covalent catalysis. Note the fact that the ring N can stablize the transition state. As is often the
case, a coenzyme N is positively charged and can serve as an “electron sink”.
Pyridoxal phosphate (PP)
A)
involved in oxidation reduction reactions
involved in reactions of amino acids
There is a family referred to as the vitamin B6 family, this includes pyridoxine, pyridoxal and
pyridoxamine. The pyridoxal (aldehyde) form is the active form and must be phosphorylated
(using ATP) before it is functional, it is then called pyridoxal phosphate.
Pyridoxal phosphate is covalently linked to the enzyme via Schiff’s base formation between the
aldehyde of PP and a lysine R group of the enzyme.
The Schiff’s base between the enzyme and PP is displaced when a substrate comes along and
there is instead a Schiff’s base formation between the PP and the α amino group of the amino
acid substrate.
The (+) N of the pyridoxal ring acts as an electron sink, destabilizing the bonds between the
main C of the amino acid and the substituent groups. Even though the N is at the bottom of the
ring, there is a conjugated double bond system that allows the electrons to move throughout the
E)
8)
Biotin
A)
B)
C)
D)
9)
C)
D)
E)
F)
D)
involved in 1 carbon transfers
it does not transfer C at the oxidation level of the carbonyl, that is done
by biotin
with one exception, it does not transfer C at the oxidation level of the
methyl group, that is done by SAM
Folate must be provided in the diet
Folate must be reduced (two different double bonds are reduced to single bonds using
NADPH), before it is active. One it is reduced, it is called tetrahydrofolate (THF). This is the
active form.
A glutamate tail must be added before folate is active. The glutamates are added by the
formation of an amide bond between the alpha N and the R group carboxyl group. This
requires ATP.
A carbon must be added to THF. This is the carbon that will be transferred to a substrate. The
source of this carbon is usually the serine R group.
The carbon is added to the N-5 or the N-10 atom (or is bound to both). Be able to recognize the
oxidation state of the carbon added. You don’t have to know the names of the various forms of
THF.
The only time THF acts to transfer a methyl group is to add one to homocysteine to reform
methionine. This occurs after SAM has donated the methyl group to some substrate and must
be regenerated.
B)
B)
involved in carrying acyl groups
This is an 8 carbon fatty acid with a sulfur substitution at carbons 6 and 8. These form an
intramolecular disulfide bond. This is broken when a fatty acid (acyl group) is transferred to one
of them, forming a thioester (the same as with coenzyme A)
After the acyl group is gone, the enzyme is found in the reduced form and must be regenerated
to the reduced disulfide form.
Ubiquinone
A)
involved in intramolecular rearrangements
cobalamin (vitamin B12) must be provided in the diet.
it contains a corrin ring (which is similar to the porphryn ring) and it contains Co +3
the cobalt is coordinated to 4 nitrogens of the corrin ring (like iron is coordinated to 4 nitrogens
of the heme. Another coordination site is occupied by an adenosyl group, donated by ATP.
The reaction mechanism involving this coenzyme involves a free radical intermediate. When
there is a free radical, it is easier for a rearrangement to occur in a molecule. The free radical
intermediate results in one electron remaining with Co and reducing the charge to +2 and
leaving the other lone electron on the C-5 of the adenosyl group.
Lipoic acid
A)
12)
Biotin must be provided in the diet.
ATP is required to attach a carboxyl group to an N of biotin.
Biotin is covalently linked via an amide bond to a lysine R group of the enzyme.
The carboxyl source is bicarbonate.
Adenosylcobalamin
A)
B)
C)
11)
the only coenzyme involved in carboxylation reactions
Folate
A)
B)
10)
molecule. Several reactions can occur. If the H is removed and put back in the opposite
stereochemistry, the enzyme would be a racemase. The carboxyl can be removed and it is a
decarboxylase (this is the example given). The amino group can be removed and this is a
transaminase.
Note diamine tetrahedral intermediate when Schiff’s base is being transferred from enzyme to
substrate. Note that when a N=C is cleaved by water, the C ends up with a = to O.
this a lipid soluble electron carrier, involved in oxidation-reduction reactions
As with FAD and FMN, this can do 1 electron transfers and has a quinone, a semiquinone, and
a hydroquinone form. The quinone is the oxidized form and the hydroquinone is the reduced
form.
If you have an A, you must have a B, so here is the B.