BAHAN KULIAH BIOKIMIA POWER POINT BAGIAN 1 /BIOCHEMISTRY POWER POINT LECTURES PART 1 | Karya Tulis Ilmiah

Organic reaction mechanisms

Much can be learned by studying organic

model reactions when compared to enzyme

catalyzed reactions.

1. Group transfer reactions

2. Oxidations and reductions

3. Eliminations, isomerizations and rearrangements

ATP

ATP is the energy carrier for most biological

reactions

ATP + H

₂

O -> ADP + P

i

Nucleophiles

R NH₂ + H+ R N+ H H

H

R NH_{2 O} R'

R''

R N C OH

R'' R' H

Basic reaction of amine

R NH_{2 O} R'

R''

R N C OH

R'' R' H R N H _R' R'' +

Amine Ketone or aldehyde

Carbinolamine intermediate

Imine

Group transfer reactions

Acetyl group transfer

Nucleophile attack on an acyl carbonyl to form a tetrahedral intermediate

Peptide bond hydrolysis Phosphoryl group transfer

nucleophile attack on a phosphate to yield a trigonal bipyramid intermediate

Kinase reactions involving transfer of phosphate from ATP to organic alcohols

Glycosyl group transfers

substitution of one group at the C1 carbon of a sugar for another

Thioesters (Acetyl-coenzyme A)

High energy compound

Carrier of acetyl and acyl groups Can be used to drive

Oxidations and reductions

Oxidation : Loss of Electrons

Reduction: Gain of Electrons

Many redox reactions involve the breaking of a C-H bond and the loss of two bonding electrons

Y + H O C

Electron transfer reactions to oxygen undergo transfer of

one electron at a time (Pauli exclusion principle)

Electron transfer reactions

B

A

B

A

red red

n 







n_ox

Half-cell reactions either donate or accept electrons

Work is non -pressure volume work or



G = -w’ = -w

elec

W

elec

= nF



E

or



G = -nF



E











 

























red

n

ox

n

ox

red

o

B

B

A

A

ln

nF

RT

-E

E

F = Faraday constant = 96,485 Coulombs per mole of electrons E0 = standard reduction potential or midpoint potential

Measuring potentials

red -n ox red -n

ox

ne

A

and

B

ne

B

A

























n

ox

red

0

A

A

A

A

Ln

nF

RT

-E

E







oe -donor



o

acceptor

-e

o

E

-

E

E



However, there is no absolute potential to reference!

(g)

H

2e

2H





-



₂

At equilibrium and in contact with a platinum electrode and at 1 M H+ _{and STP this is defined as zero potential. At pH of 7.0 this is}

-0.421 V = Eo´. Prime means that it is at pH 7.0.

Every thing is referenced to this potential

Metabolic pathways are irreversible

They have large negative free energy changes to

prevent them running at equilibrium

.

If two pathways are interconvertible (from 1 to 2

or 2 to 1), the two pathways must be different!

1

A

2

X

Y

Independent routes means

independent control of

rates.

Every pathway has a first committed step

A committed step is an irreversible step that commits

the pathway to the synthesis of the end product. This

step is usually the regulated step in the pathway.

All metabolic pathways are regulated

Pathways in eukaryotic cells occur in

separate organelles or cellular locations

ATP is made in the mitochondria and used in the

cytosol. Fatty acids are make in the cytosol and

broken down in the mitochondria. Separation of

pathways exerts a greater control over opposing

Experimental approaches to study

metabolism

1. Sequence of reactions by which a nutrient is converted to end products

2. Mechanism by which an intermediate is turned into its successor.

3. Regulation of the flow of metabolites in a pathway.

Inhibitors and growth studies are used to see what is blocked. If a reaction pathway is inhibited products before the block increase and intermediates after the block decrease in

Glycolysis

The conversion of glucose to pyruvate to yield 2ATP

molecules

•

10 enzymatic steps

•

Chemical interconversion steps

•

Mechanisms of enzyme conversion and intermediates

•

Energetics of conversions

Historical perspective

Winemaking and baking industries

1854-1865 Louis Pasture established that microorganisms were responsible for fermentation.

1897 Eduard Buchner- cell free extracts carried out fermentation no “vital force” and put fermentation in the province of chemistry

1905 - 1910 Arthur Harden and William Young

• inorganic phosphate was required ie. fructose-1,6-bisphosphate

Inhibitors were used. Reagents are found that

inhibit the production of pathway products, thereby

causing the buildup of metabolites that can be

identified as pathway intermediates.

Fluoride- leads to the buildup of 3-phosphoglycerate

and 2-phosphoglycerate

Pathway overview

1. Add phosphoryl groups to activate glucose.

2. Convert the phosphorylated intermediates into high energy phosphate compounds.

3. Couple the transfer of the phosphate to ADP to form ATP.

Stage I A preparatory stage in which glucose is phosphorylated and cleaved to yield two molecules of

glyceraldehyde-3-phosphate - uses two ATPs

Stage II glyceraldehyde-3-phosphate is converted to pyruvate with the concomitant generation of four ATPs-net profit is

2ATPs per glucose.

Oxidizing power of NAD+ must be recycled

1. Under anaerobic conditions in muscle NADH

reduces pyruvate to lactate (homolactic fermentation).

2. Under anaerobic conditions in yeast, pyruvate is

decarboxylated to yield CO

₂

and acetaldehyde and the

latter is reduced by NADH to ethanol and NAD+ is

regenerated (alcoholic fermentation).

3. Under aerobic conditions, the mitochondrial

oxidation of each NADH to NAD+ yields three ATPs

Hexokinase

Isozymes: Enzymes that catalyze the same reaction but

are different in their kinetic behavior

Tissue specific

Glucokinase- Liver controls blood glucose levels.

Hexokinase in muscle - allosteric inhibition by ATP

Hexokinase in brain - NO allosteric inhibition by ATP

O OH H OH OH H

CH₂OH H H OH H O H OH H OH H OH H OH

CH2OPO3

2-H

+ ATP + ADP + H+

Glucose Glucose-6-phosphate

Hexokinase reaction mechanism is

RANDOM Bi-Bi

Glucose ATP ADP Glu-6-PO

₄

When ATP binds to hexokinase without glucose it does not

hydrolyze ATP. WHY?

The enzyme movement places the ATP in close

proximity to C

₆

H

₂

OH group of glucose and excludes

water from the active site.

There is a 40,000 fold

increase in ATP hydrolysis

upon binding xylose which

cannot be phosphorylated!

O OH H OH OH H H H

Yeast hexokinase, two lobes are gray and green.

Binding of glucose (purple) causes a large

conformational change. A substrate induced

Phosphoglucose Isomerase

Uses an “ ene dione intermediate

1) Substrate binding

2) Acid attack by H

₂

N-Lys opens the ring

3) Base unprotonated Glu abstracts proton from C2

4) Proton exchange

5) Ring closure

OH CH₂OH

H

OH H

H O -2_O

3POCH2

HO O H OH H OH OH H OH

CH₂OPO₃

2-H

Phosphofructokinase

Fructose-6-PO

₄

Fructose-1,6-bisphosphate

1.) Rate limiting step in glycolysis

2.) Irreversible step, can not go the other way

3.) The control point for glycolysis

OH CH₂OH

H

OH H

H O -2_O

3POCH2

HO

OH

CH2OPO3-2

H

OH H

H O -2_O

3POCH2

HO

+ ATP Mg + ADP

Aldolase

CH2OPO3-2 C O C C C H HO OH H OH H

CH₂OPO₃-2

CH₂OPO₃-2 C O C H HO H C OH H

CH₂OPO₃-2

H O

+

Dihydroxyacetone phosphate (DHAP) Glyceraldehyde-3-phosphate (GAP) Fructose -1,6-bisphosphate (FBP)

There are two classes of Aldolases

Class I animals and plants - Schiff base intermediate

Step 1 Substrate binding

Step 2 FBP carbonyl groups reacts with amino LYS to

form iminium cation (Schiff base)

Step 3. C3-C4 bond cleavage resulting enamine and

release of GAP

Step 4 protonation of the enamine to a iminium cation

Step 5 Hydrolysis of iminium cation to release DHAP

C14 NH

CH2OH CH2OPO3-2

(CH₂)₄ Lys C14 NH₃

CH₂OH CH₂OPO₃-2

(CH₂)₄ Lys

H

Class II enzymes are found in fungi and algae and

do not form a Schiff base. A divalent cation usually

a Zn

+2

polarizes the carbonyl intermediate

_.

Probably the occurrence of two classes is a metabolic

redundancy that many higher organisms replaced

with the better mechanism.

CH2OPO3-2 C O

C HO

H

Zn2+

-

HO H

C O Zn2+ CH₂OPO₃

-Aldolase is very stereospecific

When condensing DHAP with GAP four possible

products can form depending on the whether the

pro

-S or

pro

R hydrogen is removed on the C3 of DHAP

and whether the

re

or

si

face of GAP is attacked.

CH₂OPO₃

2-H HO OH H OH H

CH₂OPO₃ 2-O

CH₂OPO₃

2-OH H OH H OH H

CH₂OPO₃ 2-O

D-Fructose 1,6 bisphosphate

D-Psicose 1,6 bisphosphate

CH2OPO3

2-H HO H HO OH H

CH₂OPO₃ 2-O

CH₂OPO₃

2-OH H H HO OH H

CH₂OPO₃ 2-O

D-Tagatose 1,6 bisphosphate

Triosephosphate isomerase

DHAP GAP









96

1

10

x

7

.

4

DHAP

GAP

K

eq







2



TIM is a perfect enzyme which its rate is diffusion

controlled.

TIM has an enediol intermediate

Transition state analogues Phosphoglycohydroxamate (A) and 2-phosphoglycolate (B) bind to TIM 155 and 100 times stronger than GAP of DHAP

H

C

O

CH₂OPO₃ 2-OH

H _C

CH₂OPO₃ 2-OH

H

C C

CH2OPO3

2-O

OH

H

H H OH

GAP enediol DHAP

CH2OPO3 2-O

-N OH

C

O₃2-POH₂C

O -H

HO

O₃2-POH₂C O -O

TIM has an extended “low barrier”

hydrogen bond transition state

Geometry of the eneolate intermediate

prevents formation of methyl glyoxal