Cofactors

Chemical species that participate in catalysis of enzymatic reactions.

Cofactors are required by inactive apoenzymes to convert them to active holoenzymes.
Type of cofactors:

Some enzymes containing or requiring inorganic elements as cofactors

Some coenzymes serving as transient carriers of specific atoms or functional groups

Major coenzymes
 Phosphate hydrolysis
Hydrolysis reactions for some biochemically important phosphate compounds.
The labile phosphate group of each compound is shown in yellow. The more stable reaction product,
Pi is in gray. A scale of phosphate transfer potential is shown to the right.

ATP products
Hydrolysis of ATP
Formation of S-adenosylmethionine
 NADÅ and NADPÅ
Formulas:

Oxidized and reduced from of NAD (and NADP)

The pyridine ring of NAD + is reduced by addition of a hydride ion to C-4 when NAD + is converted to
NADH (and when NADP+ is converted to NADPH). In NADP, the 2'-hydroxyl group of the sugar ring
of adenosine is phosphorylated.The reactive center of these coenzymes is shown in red.
Some reactions catalyzed by NAD +
The UV absorption spectra of NAD + and NADH.

Reduction of the nicotinamide ring produces a new, broad absorption band with a maximum at 340
nm. The production of NADH during an enzyme-catalyzed oxidation can be conveniently followed by
observing the appearence of the absorbaance at 340 nm.
Lactate dehydrogenase - a NAD-dependent enzyme

Lactate dehydrogenaze catalyses the reversible oxidation of lactate

Mechanism of lactate dehydrogenase.

His-195, a base catalyst in the active site, abstracts a proton from the C-2 hydroxyl group of lactate,
facilitating transfer of the hydride ion (H -) from C-2 of the substrate to C-4 of the bound NAD +.
Because lactate dehydrogenase has a specific binding site for the carboxamide group (-C(O)-NH 2) of
NAD +, only one face of the pyridine ring is positioned toward lactate and therefore the hydride ion is
always added to the same face. Arg-171 forms an ion pair with the carboxylate group of the substrate.
In the reverse reaction, H - is transferred stereo-specifically from the reduced coenzyme, NADH, to C-
2 of the oxidized substrate, pyruvate.
Ordered kinetic mechanism for lactate dehydrogenase

In the forward direction, the coenzyme NAD + is bound first and its reduced form, NADH, is released
last.

FAD and FMN

FAD: flavin adeninc dinucleotide
FMN: flavin mononucleotide
both are derived from ribofiavin, or vitamin B2
Riboflavin consist of the five-carbon alcohol ribitol (a reduced form of ribose) linked to a nitrogen
atom of isoalloxazine, a heterocyclic rings system.
Like NAP+ and NADP+, FAD contains AMP and a pyrophosphatc linkage.
Riboflavin and its coenzymes

Riboflaivin. Ribitol is linked to the isoalloxazine ring system.

Flavin mononucleotide (FMN; black) and flavin adenine dinucleotide (FAD; black and blue). The
reactive center is shown in red.
Reduction and reoxidation of FMN or FAD

The conjugated double bonds between N-1 and N-5 are reduced by addition of a hydride ion and a
proton to form FMNH 2 or FADH 2, respectively, the hydroquinone form of each coenzyme. Oxidation
occurs in two steps. A single electron is removed by a one-electron oxidizing agent, with loss of a
proton, to form a relatively stable free-radical intermediate. This semiquinone is then oxidized by
removal of a proton and an electron to form fully oxidized FMN or FAD.

Reactions calatyzed by flavoproteins

Mechanism of the flavin-dependent glutathione reductase reaction.

The first steps, not shown, involve the reduction of FAD to FADH 2 by NADPH and the binding of
glutathione (glutathione is a sulfhydryl compound). The mechanism by which oxidized glutathione is
reduced by the E. FADH 2 is shown.

Vitamins and vitaminlike nutrients

Major vitamins required in human nutrition
 Niacin

Pellagra
A clinical deficiency syndrome manifested in skin, nervous system, and digestive tract due to
deficiency of niacin.
Lesion of skin exposed to light
Spinal pain, digestve disturbance

Vitamin B1 (Thiamine)

Structure of thiamine

Mechanism of thiamine pyrophosphate action

Mechanism of yeast pyruvate decarboxylase
The positive charge of the thiazolium ring of TPP attracts electrons, weakening the bond between C-
2 and hydrogen. This proton is presumably removed by a basic residue. Ionization generates a
resonance-stabilized dipolar carbanion known as an ylid (a molecule with opposite charges on
adjacent atoms). The negatively charged C-2 attacks the electron-deficient carbonyl carbon of the
substrate pyruvate, and the first product (CO2) is released. Two carbons of pyruvate are now attached
to the thiazole ring as part of a resonance-stabilized carbanion. In the following step, protonation of
the carbanion produces hydroxyethylthiamine pyrophosphate (HETPP). HETPP is cleaved,
releasing acetaldehyde (the second product) and regenerating the ylid form of the enzyme-TPP
complex. TPP re-forms when the ylid is protonated by the enzyme.

Viatmin B2 (Riboflavin)
 Vitamin B6 (Pyridoxine)

B 6 vitamins and pyridoxal phosphate

 Pyridoxal phosphate
Binding of PLP

The fundamental biochemical function of pyridoxal-5-phosphate is the formation of aldimines with a-

amino acids that stabilize the development of carbanionic character at the a and bcarbons of a-amino
acids in intermediates.

Structures of catalytic intermediates in pyridoxal-phosphate-dependent reactions.

The initial aldimine intermediate resulting from Schiff's base formation between the coenzyme and
the a-amino group of an amino acid (a). This aldimine is converted to the resonance-stabilized
intermediate (b) by loss of a proton at the a carbon. Further enzyme-catalyzed proton transfers to
intermediates (c) and (d) may occur, depending on the specificity of a given enzyme. The enzymes
use their general acids and bases to catalyze these proton transfers.

Mechanism of transaminases
 Pyridoxal phosphate (continued)
Some important metabolic roles of pyridoxal phosphate

Mechanism of action of pyridoxal phosphate in glutamate oxalacetate transaminase

Mechanism of action of pyridoxal phosphate in aspartate b-decarboxylase

Tetrahydrofolate
Formation of tetrahydrofolate
Pterin is part of folate, a molecule containing p-aminobenzoate (red) and glutamate (blue). The
polyglutamate forms of tetrahydrofolate usually contain five or six glutamate residues. The reactive
centers of the coenzyme, N-5 and N-10 are shown in red.

One-carbon derivatives of tetrahydrofolate

The derivatives can be interconverted enzymatically by the routes shown.
R represents the benzoyl polyglutamate portion on tetrahydrofolate

Vitamin B12 (Cobalamin)

Metabolism of vitamin B 12
The metabolic interconversions of B12 are indicated with light arrows, and B12 requiring reactions are
indicated with heavy arrows. Other related pathways are indicated with dashed arrows.

Biotin
Biotin is a cofactor for enzymes that catalyze carboxyl-group-transfer reactions and ATP-dependent
carboxylation reactions.

The carboxylate group of biotin is covalently bound via amide linkage to the e-amino group of a
lysine residue (blue). The reactive center of the biotin moiety is N-1 (red).

Reaction catalyzed by pyruvate carboxylase

 Biotin, bicarbonate and ATP react to form carboxybiotin
Carboxybiotinyl-enzyme complex provides a stable activated form of CO2 that can be transfererd to
pyruvate
The enolate form of pyruvate attacks the carboxyl group of carboxybiotin forming oxaloacetate and
regenerating biotin.

Coenzyme A

Vitamin C
Ascorbate metabolizing pathways and their reagulation by stress in animal cells

Physiological and experimental agents affecting ascorbate metabolism are shown in italics; the most important
mediators of stress response are underlined.
Tocopherol recycling by ascorbate

RBC: human erythrocyte

ASC:ascorbate

T: alpha-tocopherol

X: free radical species

Recycling of lipid peroxyl radicals by alpha-tocopherol, and of alpha-tocopherol by ascorbate

Biosynthessis and assembly of collagen
 Step 1-4 occurs in the cytoplasm of collagen-synthetizing cells
Steps 6 amd 7 occur in the extracellural region
Gal = galactose
Glc = glucose

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