Che 214 lecture 03

CHE 214: Biochemistry

Lecture Three

•NUCLEIC ACIDS•BIOENERGETICS

Lecturer: Dr. G. Kattam Maiyoh

GKM/CHE 214/LEC 03/SEM 02/2013

d. Nucleic Acids • DNA –deoxyribonucleic acid– Polymer of deoxyribonucleotide triphosphate (dNTP) – 4 types of dNTP (ATP, CTP, TTP, GTP)– All made of a base + sugar + triphosphate

• RNA –ribonucleic acid – Polymer of ribonucleotide triphosphates (NTP)– 4 types of NTP (ATP, CTP, UTP, GTP)– All made of a base + sugar + triphosphate

• So what’s the difference? – The sugar (ribose vs. deoxyribose) and one base (UTP vs.

TTP)


Deoxyribose (like ribose) is a sugar with 5 carbon atoms in a ringOxygen is one of the ring members

In Deoxyribose, one of the OH groups is missing and replaced with hydrogen, Thus deoxy = - 1 oxygen

Phosphate groups are important because they link the sugar on one nucleotide onto the phosphate of the next nucleotide to make a polynucleotide.


• Nitrogenous bases

• In DNA the four bases are:– Thymine– Adenine– Cytosine– Guanine

• In RNA the four bases are:– Uracil– Adenine– Cytosine– Guanine

Base - pairing


DNA and RNA are polynucleotides

• Both DNA and RNA are polynucleotides.• They are made up of smaller molecules

called nucleotides.• DNA is made of two polynucleotide strands:

• RNA is made of a single polynucleotide strand:

Nucleotide NucleotideNucleotide

Nucleotide

Nucleotide

Nucleotide

Nucleotide

Nucleotide

Nucleotide Nucleotide Nucleotide Nucleotide

NucleotideNucleotide Nucleotide

Nucleotide


• Nucleic Acids Function – Information Storage • DNA / mRNA

– Information transfer / Recognition • rRNA / tRNA / snRNA

– Regulatory • microRNA ?


DNA •Information for all proteins stored in DNAin the form of chromosomes or plasmids. •Chromosomes (both circular and linear) consist of two strands of DNA wrapped together in a left handed helix.(imagine screwing inwards)

•The strands of the helix are held together by hydrogen bonds between the individual bases. •The “outside” of the helix consists of sugar and phosphate groups, giving the DNAmolecule a negative charge. GKM/CHE 214/LEC 03/SEM 02/2013


The Rule: Complimentarity• Adenine always base pairs with Thymine (or

Uracil if RNA)

• Cytosine always base pairs with Guanine.

• This is because there is only exactly enough room for one purine and one pyrimidine base between the two polynucleotide strands of DNA/RNA. These bases are complimentary to each other


Complimentary Base Pairs

A-T Base pairing G-C Base Pairing



DNA Structure

• The DNA helix is “anti-parallel” – Each strand of the helix

has a 5’ (5 prime) end and

a 3’ (3 prime) end.


DNA Structure

Strand 1

(Watson strand)

Strand 2 (Crick strand)

5 ‘ end

3 ‘ end

3’ end

5’end


DNA Structure

• 1 atgatgagtg gcacaggaaa cgtttcctcg atgctccaca gctatagcgc caacatacag • 61 cacaacgatg gctctccgga cttggattta ctagaatcag aattactgga tattgctctg • 121 ctcaactctg ggtcctctct gcaagaccct ggtttattga gtctgaacca agagaaaatg• 181 ataacagcag gtactactac accaggtaag gaagatgaag gggagctcag ggatgacatc• 241 gcatctttgc aaggattgct tgatcgacac gttcaatttg gcagaaagct acctctgagg • 301 acgccatacg cgaatccact ggattttatc aacattaacc cgcagtccct tccattgtct• 361 ctagaaatta ttgggttgcc gaaggtttct agggtggaaa ctcagatgaa gctgagtttt • 421 cggattagaa acgcacatgc aagaaaaaac ttctttattc atctgccctc tgattgtata

Because of the base pairing rules, if we know one strand we also know what the other strand is. Convention is to right from 5’ to 3’ with 5’ on the left.


Chromosomes and Plasmids• Chromosomes are composed of DNA and

proteins. – Proteins (histone & histone like proteins) serve a

structural role to compact the chromosome. – Chromosomes can be circular, or linear.• Both types contain an antiparallel double helix!

– Genes are regions within a chromosome. • Like words within a sentence.


Region (red box) of chromosome XI from the bakers yeast S. cerevisiae.Red and Blue colored boxes are genesNote that either strand may encode a gene, but that all genes start at the 5’ end and finish at the 3’ end.

http://www.yeastgenome.org/


RNA• Almost all single stranded (exception is RNAi).• In some RNA molecules (tRNA) many of the

bases are modified (e.g. psudouridine).• Has capacity for enzymatic function

-ribozymes• One school of thought holds that early

organisms were based on RNA instead of DNA (RNA world).


RNA

• Several different “types” which reflect different functions– mRNA (messenger RNA)– tRNA (transfer RNA)– rRNA (ribosomal RNA)– snRNA (small nuclear RNA) – RNAi (RNA interference)


RNA function• mRNA – transfers information from DNA to

ribosome (site where proteins are made)• tRNA – “decodes” genetic code in mRNA, inserts

correct A.A. in response to genetic code.• rRNA-structural component of ribosome• snRNA-involved in processing of mRNA• RNAi-double stranded RNA, may be component of

antiviral defense mechanism.


RNA

A - hairpin loop B- internal loop

C- bulge loop D- multibranched loop

E- stem F- pseudoknot

Complex secondary structures can form in linear molecule


mRNA

• Produced by RNA polymerase as product of transcription

– Provides a copy of gene sequence for use in translation (protein synthesis).

– Transcriptional regulation is major regulatory point – Processing of RNA transcripts occurs in eukaryotes• Splicing, capping, poly A addition

– In prokaryotes coupled transcription and translation can occur


The Central Dogma of molecular Biology


Bioenergetics


•It is the study of the energy relationships and energy conversions in biological systems.

•All organisms need free energy to keep themselves alive and functioning.

•The source of energy is just one; solar energy.

•Only plants use that energy directly.

•What the other organisms use is the chemical energy in the form of foods.

•The very first conversion of solar energy into a chemical energy is the sugar molecule.

What is Bioenergetics ?


Respiration• Respiration is important for bioenergetics

as it stores the energy to form a molecule ATP; adenosine triphosphate.

• This molecule is a link between catabolism and anabolism.

• The process of photosynthesis is helpful in understanding the principles of energy conversion i.e. bioenergetics.


• Metabolism refers to all the chemical reactions of the body– some reactions produce the energy stored in

ATP that other reactions consume– all biological molecules will eventually be

broken down and recycled or excreted from the body


25-27

Catabolism and Anabolism• Catabolic reactions breakdown complex

organic compounds– providing energy (exergonic)

– glycolysis, Krebs cycle and electron transport

• Anabolic reactions synthesize complex molecules from small molecules – requiring energy (endergonic)

• Exchange of energy requires use of ATP (adenosine triphosphate) molecule.


GKM/CHE 214/LEC 03/SEM 02/201125-28

ATP Molecule & Energy

• Each cell has about 1 billion ATP molecules that last for less than one minute

• Over half of the energy released from ATP is converted to heat

a

b


25-29

Mechanisms of ATP Generation

• Phosphorylation is the addition of phospahate group.– bond attaching 3rd phosphate group contains stored

energy • Mechanisms of phosphorylation

– within animals• substrate-level phosphorylation in cytosol• oxidative phosphorylation in mitochondria

– in chlorophyll-containing plants or bacteria• photophosphorylation.



Phosphorylation in Animal Cells

• In cytoplasm (1)• In mitochondria (2, 3 & 4)

25-30GKM/CHE 214/LEC 03/SEM 02/2013

25-31

Carbohydrate Metabolism--In Review• In GI tract

– polysaccharides broken down into simple sugars – absorption of simple sugars (glucose, fructose &

galactose)

• In liver – fructose & galactose transformed into glucose– storage of glycogen (also in muscle)

• In body cells --functions of glucose– oxidized to produce energy– conversion into something else– storage energy as triglyceride in fat


25-32

Fate of Glucosei. ATP production during cell respiration

– uses glucose preferentially

i. Converted to one of several amino acids in many different cells throughout the body

ii. Glycogenesis– hundreds of glucose molecules combined to form

glycogen for storage in liver & skeletal muscles

i. Lipogenesis (triglyceride synthesis)– converted to glycerol & fatty acids within liver & sent to

fat cells


GKM/CHE 214/LEC 03/SEM 02/201125-33

Glucose Movement into Cells• In GI tract and kidney tubules,

Na+/glucose symporters• Most other cells, GluT facilitated

diffusion transporters move glucose into cells– insulin increases number of GluT

transporters in the membrane of most cells

– in liver & brain, always lots of GluT transporters

• Glucose 6-phosphate forms immediately inside cell (requires ATP) thus, glucose hidden in cell

• Concentration gradient favorable for more glucose to enter


GKM/CHE 214/LEC 03/SEM 02/201125-34

Glucose Catabolism

• Cellular respiration– 4 steps are involved

– glucose + O2 producesH2O + energy + CO2

• Anaerobic respiration– called glycolysis (1)

– Results in formation of acetyl CoA (2)is transitional step to Krebs cycle

• Aerobic respiration– Krebs cycle (3) and electron transport chain (4)


Historical PerspectiveGlycolysis was the very first biochemistry or oldest biochemistry studied.It is the first metabolic pathway discovered.

Louis Pasture 1854-1864: Fermentation is caused by microorganism. Pastuer’s effect: Aerobic growth requires less glucose than anaerobic condition.

Buchner; 1897: Reactions of glycolysis can be carried out in cell-free yeast extract.

Harden and Young 1905: 1: inorganic phosphate is required for fermentation. 2: yeast extract could be separated in small molecular weight essential coenzymes or what they called Co-zymase and bigger molecules called enzymes or zymase.

Inhibitor studies: Iodoacetate treatment resulted in the accumulation of fructose 1,6biphosphate. Similarly fluoride caused accumulation of 2-phosphoglycerate and 3-phosphoglycerate.

1940: with the efforts of many workers, complete pathways for glycolysis was established.

Louis Pasteur (1822-1895)

Glycolysis takes place in the cytosol of cells.

Glucose enters the Glycolysis pathway by conversion to glucose-6-phosphate.

Initially there is energy input corresponding to cleavage of two ~P bonds of ATP.

H O

OH

H

OHH

OH

CH2OPO32−

H

OH

H

1

6

5

4

3 2

glucose-6-phosphate

H O

OH

H

OHH

OH

CH2OH

H

OH

H H O

OH

H

OHH

OH

CH2OPO32−

H

OH

H

23

4

5

6

1 1

6

5

4

3 2

ATP ADP

Mg2+

glucose glucose-6-phosphate

Hexokinase

1. Hexokinase catalyzes:

Glucose + ATP glucose-6-P + ADP

The reaction involves nucleophilic attack of the C6 hydroxyl O of glucose on P of the terminal phosphate of ATP.

ATP binds to the enzyme as a complex with Mg++.

GKM/CHE 214/LEC 03/SEM 02/201125-39

Glycolysis of Glucose & Fate of Pyruvic Acid• Breakdown of six-carbon glucose molecule into

2 three-carbon molecules of pyruvic acid

– 10 step process occurring in cell cytosol

– produces 4 molecules of ATP after input of 2 ATP

– utilizes 2 NAD+ molecules as hydrogen acceptors

• If O2 shortage in a cell

– pyruvic acid is reduced to lactic acid so that NAD+ will be still available for further glycolysis

– Lactic acid rapidly diffuses out of cell to blood

– Liver cells remove it from blood & convert it back to pyruvic acid


GKM/CHE 214/LEC 03/SEM 02/2011 25-40

10 Steps of Glycolysis



25-42

Formation of Acetyl Coenzyme A• Pyruvic acid enters the

mitochondria with help of transporter protein

• Decarboxylation– pyruvate dehydrogenase converts 3

carbon pyruvic acid to 2 carbon fragment (CO2 produced)

– pyruvic acid is oxidized so that NAD+ becomes NADH

• 2 carbon fragment (acetyl group) is attached to Coenzyme A to form Acetyl coenzyme A which enter Krebs cycle– coenzyme A is derived from

pantothenic acid (B vitamin).




Krebs Cycle (Citric Acid Cycle)• Series of oxidation-

reduction & decarboxylation reactions occurring in matrix of mitochondria

• It finishes the same as it starts (4C)– acetyl CoA (2C) enters at

top & combines with a 4C compound

– 2 decarboxylation reactions peel 2 carbons off again when CO2 is formed

The names of the various enzymes in the previous slide are indicated in the figure below

THE TCATHE TCA



Products of the Krebs Cycle• Energy stored in bonds is released step by step to form several

reduced coenzymes (NADH & FADH2) that store the energy

• In summary: each Acetyl CoAmolecule that enters the Krebscycle produces yields;

– 2 molecules of CO2

• one reason O2 is needed

– 3 molecules of NADH + H+

– one molecule of ATP

– one molecule of FADH2

• Remember, each glucoseproduced 2 acetyl CoA molecules



The Electron Transport Chain

• Involves a series of integral membrane proteins in the inner mitochondrial membrane capable of oxidation/reduction

• Each electron carrier is reduced as it picks up electrons and is oxidized as it gives up electrons

• Small amounts of energy is released in small steps

• Energy used to form ATP by chemiosmosis


Chemiosmosis• Small amounts of energy

released as substances are passed along inner membrane

• Energy used to pump H+ ions from matrix into space between inner & outer membrane

• High concentration of H+ is maintained outside of inner membrane

• ATP synthesis occurs as H+ diffuses through a special H+ channel in inner membrane


Steps in Electron Transport

• Carriers of electron transport chain are clustered into 3 complexes that each act as proton pump (expel H+)

• Mobile shuttles pass electrons between complexes• Last complex passes its electrons (2H+) to a half of O2 molecule to

form a water molecule (H2O)


Proton Motive Force & Chemiosmosis

• Buildup of H+ outside the inner membrane creates + charge– electrochemical gradient potential energy is called proton motive force

• ATP synthase enzyme within H+ channel uses proton motive force to synthesize ATP from ADP and P


Summary of Cellular Respiration• Glucose + O2 is broken down into CO2

+ H2O + energy used to form 36 to 38 ATPs– 2 ATP are formed during glycolysis – 2 ATP are formed by phosphorylation

during Krebs cycle– electron transfers in transport chain

generate 32 or 34 ATPs from one glucose molecule

• Points to remember – ATP must be transported out of

mitochondria in exchange for ADP• uses up some of proton motive force

– Oxygen is required or many of these steps can not occur

Documents

Che 214 lecture 03