Biochemistry-Midterm Flashcards
Octamer complex
H2A, H2B, H3, and H4 dimers
H1 protein
Binds to the 30 bp linker so that the DNA doesn’t become a mess
Denaturation of DNA is at…
95 degrees Celsius
Renaturation of DNA is at…
37 degrees Celsius
Melting temperature
Temperature at which half of the DNA is denatured
Hybridization
Uses renaturation and denaturation to see how similar the genetic material of two species is
Polymerase chain reaction (PCR)
Uses denaturation and renaturation to amplify a target sequence to make a specific protein
Introns
In between exons
Origin of replication (ori)
Full of AT rich sequences
DNAa
Binds to the ori and stretches it to break the hydrogen bonds
Helicase
Unwinds the DNA by breaking the hydrogen bonds
Single strand binding proteins (SSBP)
Binds to the unwound strands so that they won’t get back together. Also protects genetic material from being degraded by nucleases
Primase
Synthesizes the RNA primer
Integrase
Helps paste the DNA transposon to the new location
Topoisomerase I
Relieves stress of the supercooled DNA on one strand by cutting, unwinding, and resealing
Topoisomerase II
Helps relieve stress on the supercoiled DNA on both strands by cutting, unwinding, and resealing
DNA polymerase III
A holoenzyme consisting of 10 subunits. Includes:
5’-3’ polymerase
3’-5’ exonuclease
5’-3’ polymerase
Adds nucleotides to the growing strand
3’-5’ exonuclease
Proofreads DNA to check for errors
Leading strand
Needs one RNA primer and goes towards the replication fork
Lagging strand
Needs multiple RNA primers and goes way from the replication fork. Segments are discontinuous and are called Okazaki fragments
Replication bubble/fork
Y-shaped opening that opens up the DNA to being replication
DNA polymerase I
Works on the lagging strand after DNA polymerase III adds nucleotides
5’-3’ exonuclease
Gets rid of the RNA primers on the lagging strand
Ligase
Joins the discontinuous fragments
Polymerase alpha
Primase in eukaryotes
Polymerase epsilon
Synthesizes the leading strand and fills in the gaps in between the primers on the lagging strand
Polymerase delta
Synthesizes the lagging strand
RNase and flap endonuclease 1 (FEN1)
Removes the primers
Tandem (satellite)
Repeated sequences right next to each other
Interspersed (transposons)
Respected at different sections of the DNA
Telomeres don’t shorten in…
Cancer, germ, and stem cells
Didanosine
Analog of adenine that fails to form a phosphodiester bond since it has H instead of OH. HIV/AIDs drug
Azidothymidine (AZT)
Analog of thymine that has an acidosis group instead of an OH. Doesn’t form a phosphodiester bond and is and HIV/AIDs drug
Camptothecin (CPT)
Inhibits topoisomerase I from binding to its site. A cancer drug
Eptoposide
Topoisomerase II inhibitor and a cancer drug
DNA polymerase proofreading errors
Mutations in the 3’ to 5’ exonuclease
Depurination
Removing the purines and so 3’-5’ skips over it. Results in deletion mutation
Deamination
Removal of amine group. Transition mutation.
Cytosine-> uracil
Guanine-> xanthine
Adenine-> hypo-xanthine
5’methylcytosine-> thymine
Transition mutation
Changing from purine to purine or pyrimidine to pyrimidine
Transversion mutation
Changing from purine to pyrimidine
ROS
ROS binds to DNA and damages it. 8-hydroxyl guanosine changes to thymine
Thymine dimers
Thymines next to each other bind and leads to structural damage
Point mutation
Change in one nucleotide
Structural damage
Breaks backbone
Base excision repair
Fixes deamination
Nucleotide excision repair
Fixes thymine diners
Mismatch repair
Fixes misincorporated bases. Uses MutS, MutL, and MutH
Missense mutation
Changes nucleotide leading to different amino acid
Nonsense mutation
Puts a premature stop codon
Frame shift mutation
Either an insertion or deletion
Silent mutation
Change in nucleotide but no change in amino acid coded
Xeroderma pigmantosium
Autosomal recessive condition where people are photosensitive. Results in mutation in nucleotide excision repair which corrects for thymine dimers
Ataxia-telangiectasia
Mutation in ATM gene which tells that there is an issue. Due to no correction in ROS. Degenerative motor condition due to failure to repair ROS in the cerebellum. Can’t call base excision repair system
Hereditary non-polyposis colon cancer (HNPCC)
Mutation in mismatch repair system
Restriction enzymes (endonucleases)
Cleave specific DNA sequences
Staggered cuts
Produces sticky ends/cohesive ends that have H bonds. Ligand can connect them together
Blunt cuts
Produce blunt ends that don’t have H bonds. Enzyme in the T4 bacteriophage helps ligase strands together
Gel electrophoresis
Electrical field in which DNA goes to the positive side (phosphates of DNA) make it negative. Separated molecules based on size
Requirements of PCR
Taq DNA polymerase
Two DNA primers
dNTP
Template DNA
3 steps of PCR
Denaturation
Annealing
Extension
Applications of PCR
Disease diagnosis
Disease identification
Treatment
DNA sequencing
Melting temperature
Tm= 2(# of A&T) + 4(# of G&C)
DNA sequencing
Finding out the sequencing of DNA. Two steps:
Generating the sequence
Obtaining the sequence
Maxam Gilbert method
Chemical process that has a lot of limitations
Sanger’s method
Enzymatic, dNTP, ddNTP, electrophoresis
Pyrosequencing
Enzymatic, dNTP, no ddNTP, no electrophoresis
ddNTP color
ddATP: green
ddGTP: black
ddCTP: blue
ddTTP: red
DNA sequencing procedure
Put all materials in PCR and amplify for 40 cycles. Clean the sample and put in electrophoresis
Applications of DNA sequencing
Genetic mutation
Gene function and structure
DNA cloning
RNA
Working copy of the DNA
tRNA
Makes up 15% of total RNA in the cell. Smallest RNA type in the cell. Decodes the nucleotide sequence to form amino acids at the anticodon loop
rRNA
Makes up 80% of total RNA in the cell.
Four different species: 28S, 18S, 5.8S, and 5 S
Attaches to ribosomal proteins
mRNA
Most heterogenous RNA in terms of shape and structure. Makes up 3-5% of total RNA.
Has special structures:
- 5’ cap
- 5’-3’ UTR
- poly A tail at 5’ end
Heterochromatin
Condensed form of DNA. Genes are inactive. HDAC removes the acetyl group from lysine to form a strong DNA/histone bond
Euchromatin
Less condensed structure. Has active genes. HATS adds acetyl group to the lysine to form a loose DNA/histone interaction
RNA polymerase I
Transcribes pre-RNA sequences of 28S, 18S, and 5.8S
RNA polymerase II
Transcribes mRNA and ncRNA (snRNA, miRNA, scoRNA)
RNA polymerase III
Transcribes tRNA and small amounts of snoRNA and snRNA
Promoter region
Initiates/promotes transcription
DNA elements
Sequences in the promoter region. Two examples:
- TATA box: 25 nucleotides upstream (down first nucleic acid)
- CAAT box: 70-89 nucleotides upstream
Transcription factors
Binds to the DNA elements
Example: CTF1, SP1, and TFIID (binds to TATA box)
Alpha-amanatin
Toxin procure by amanita phaloides mushroom. Binds to RNA polymerase II and is irreversible.
Causes GI disturbances, electrolyte imbalance, and kidney+liver dysfunction
45 S
Pre- RNA
Pre- RNA is modified by…
- Cleavage by an endonuclease (RNase)
- Trimmed by exonucleases
- Base and nucleotide modification by snoRNA
tRNA modifications
- 16 nucleotide sequence at 5’ end is cleaved
- 14 nucleotide sequence at anticodon loop is cleaved
- 3’ end uracil residue is converted into CCA
- Nucleotide modifications
Primitive mRNA
RNA polymerase II transcribes a premature RNA called heterogenous RNA (hnRNA)
hnRNA modifications…
- Cap at 5’ end (co)
- Poly-A tail at 3’ end (post)
- Splicing (co or post)
5’ Capping
Cap is a 7-methylguanosine which is added backwards frmom 5’ to 5’. Cal is not transcribed from the DNA
Stabilizes mRNA and helps in initiating translation
Guanylyltransferase
Adds the GMP (guanosine monophosphate)
7-guonosine methyltransferase
Adds the methyl group to the guanosine
Poly- A tail
Tail is added after it recognizes the polyadenylation sequence (AAUAAA). Tail ranges from 40-200 nucleotides.
Helps in stabilizing, in translation, and transporting out of the cell
Longer poly-A tail, more stable it is since exonucleases will have to eat more to get to the actual mRNA
Poly A tail sequence is not transcribed from the DNA
Polyadenylation polymerase (poly A pol)
Catalyze the poly-A tail. ATP is used as a substrates
Splicing
Cutting introns to only ligate the exons
Intron structure
5’ end: start with GU
3’ end: start with AG
Branch point A
snRPS
Small nuclear ribonuclear proteins. They help in splicing. Contain snRNAs
Splicing mechanism
2’OH of branch point A attacks 5’ end of intro closest to exon 1. This forms a 2’ to 5’ bond and lacerates it. The free 3’ end of exon 1 then binds to the 5’ of intron closest to exon 2.
B-knot thalesimia
Homozygous mutation in the intron region (5’ or 3’ end) in the hemoglobin gene. This totally abolishes normal splicing and is fatal!
B+ thalesimia
Point mutation in polyadenylation sequence. Instead of AAUAAA -> AACAAA. Less fatal
Epigenetic change
Modification of base pairs that leads to irreversible and hereditary changes. There is no change in bases
Epigenetic changes example
- The way you hold a baby
- Cellular differentiation
- Differences in monozygotic twins
Mice example
Normal mice has brown fur, is small, and free from disease. Agouti mice has yellow fur, is large, and prone to disease.
When give mother normal diet and B12, folate, choline, and betaine, mice were normal.
When give mother normal diet only, mice were agouti
Bee example
Both queen and workers have same genes. However, queen takes royal jelly diet while worker doesn’t take this diet
DNA methylation
Silences genes
DNA methyl-tansferase
Adds methyl group to 5 part of CpG cytosine.
Can measure these levels in original gene or transcribed target sequences
5mC inhibits gene activity because…
- Harder to break hydrogen bonds
- Larger is TFs wont attach
- Strong binding with methyl proteins so won’t bind to TFs
- Chromatin remodeling
Places where you can get methyl…
- Folic acid
- Betaine
- B12
- B6
- Choline
Importance of DNA methylation
- DNA imprinting
- X chromosome inactivation
- Aging
- Tissue specialization
H2A and H2B…
Leave the cell readily so not important for gene modification
H3 and H4…
Stats in cell and is important for gene modification
Histone acetylation
Charges of arginine and lysine are suppressed so genes are active
Histone deacetylation
Charges if arginine and lysine are activated and genes are inactive
Resveratol
Found in red grapes
Removes acetyl group and improves health
Housekeeping genes
Need them all the time.
Ex: ribosomal genes, tRNA, actin
Controlled genes
Need them at specific times.
Ex:histone, DNA pol, hormones
Regulatory DNA sequences
On the DNA and are cis acting elements (come from the same gene). Can enhance/silence gene expression
Regulatory proteins (TFs)
Trans-acting DNA proteins that bind to DNA elements. That activate/suppress gene expression
Steroid hormone receptor example
Glucocorticoid receptors are separate. When cortisol binds to the receptors, they form a protein dimer. This signals the GRE sequence to come and activate certain genes
Cortisol: DNA binding protein
GRE: DNA sequence
Splice site choice
Can choose what regions you want to splice
mRNA editing
Change in a nucleotide sequence
Ex: apoprotein B’s real form is actually really long. Change in a nucleotide sequence leads to a premature stop (missense) This protein is found in the liver and small intestine
In iron deficiency…
Body thinks transferrin has iron so will increase transcription of transferrin receptors while ferritin receptor transcription will decrease
Proto-oncogene
Normal gene. Can become cancerous by accumulation of normal protein or forming an abnormal product
Oncogene
Abnormal growth in cells. Can be cause by viral insertion and cellular mechanisms
Viral insertion
When a virus enters its genetic material into the cell, it fuses with the cells’ DNA and leads the cell to make a lot of proteins
Point mutation
Leads to the change in one amino acid
Ex: in RAS a point mutation leads to a change in only one amino acid
Amplification
Usually, when genes are amplified, they are stored in the chromosome as a double minute. Amplification leads to an increase in normal gene product.
Ex: myc oncogenes is found in many neuroblastomas
Chromosomal translocation
Can lead to chimeric genes
Ex: translocation between chromosome 9 & 22 leads to a fusion protein called bcr-abl that is defective in normal protein kinase
Abl protooncogene: encodes a tyrosine specific protein kinase
src gene
Found in the Rous Sarcoma virus and is a protein kinase tyrosine (phosphorylates tyrosine). When the oncogene is activated, it will phosphorylate other proteins as well
RAS gene
Encodes guanine-nucleotide binding proteins. Activity is controlled by GTP. RAS is activates by a point mutation. When activated, it will lead to enhanced signal induction though Raf-1 serine threonine kinase, ERK-1 AND ERK-2, and induction of transcription of early genes
Ribosomes
Place where proteins are manufactured.
Have two subunits:
- Large subunit (catalytic)-cleave
- Small subunit (decoding)- reading mRNA
Have three functional sites:
- A: place where elongator aa-tRNA will bind and form the polypeptide
- P: place where initiator aa-tRNA will bind and accepts peptidyl tRNA
- E site: where the uncharged tRNA exits
Between the A and P site, there is a hole where the protein will exit from
Bacterial ribosomes
30S and 50S to make 70S
Eukaryotic ribosomes
40S and 60S to make 80S
tRNA
Has unusual bases (T). Have at least 20 tRNAs for each amino acid. Has an acceptor arm (3’ end) for carrying the amino acid and an anticodon loop for decoding the mRNA.
Required for:
- Activation or polypeptide since peptide bone formation is not feasible in normal environment
- Sequencing the amino acid in the correct order
Initiator tRNA
Transports methionine in eukaryotes and f-met in prokaryotes. This is the first amino acid and recognizes it by the sequence AUG
aa-tRNA
Charging of amino acid with the correct amino acid. Completed by aa-tRNA synthetase which is a proofreading mechanism to make sure correct tRNA and amino acid are selected. Thus activates the COOH group of the amino acid.
ATP+tRNA+Amino acid->AMP+ 2pi+ aa-tRNA
Selecting a correct amino acid
- Size
- Thermodynamicity
- Hydrophilicity or hydrophobicity
- Acceptor arm
- C:G 3:70
- Anticodon arm
Double sieve mechanism
If size (so thermodynamicity) of two amino acids are similar, need to go to charge
There are two sites in aa-tRNA
- Catalytic site: hydrophobic
- Proof reading site: hydrophilic
Amino acid must go through the hydrophobic site. If it goes though the hydrophilic site, it will be thrown out
mRNA in prokaryotes
Has 5’ to 3’ UTR. Starts are AUG codon. No introns, capping, or poly-A tail. Translation finishes at UAA, UGA, or UAG. Initiator aa-tRNA carries f-meth
mRNA in eukaryotes
Has capping, poly A tail,and splicing. Cap assembles at the ribosome but translation doesn’t start until the AUG sequence is read in the Kozak sequence. Translation stops at UAA, UAG, and UGA
Initiation
Happens in two steps:
- Assembling the 30S subunit
- Attaching the 30S subunit with the 50S subunit
Assembling the 30S subunit
- IF 1 and 3 are bound to the subunit
- IF 1 blocks the A site
- IF 3 blocks site for attaching to the 50S subunit
- IF 2 and 1 take the initiator aa-tRNA and take it to P site
- IF 2 takes the initiator t-RNA and places it at the P site
Binding the 30S subunit to 50S subunit
When the initiator aa-tRNA is attached IF 3 disconnects to allow the 50S subunit to attach
Elongation
Four steps:
- Putting elongation aa-tRNA on A site
- Formation of the peptide bond
- Moving from A to P
- Moving uncharged tRNA to E site
Have different EFs for prokaryotes and eukaryotes
- Prokaryotes: eEF-1, eEFG
- Eukaryotes: EF-Tu, EF-Ts, and EFG
Putting elongation aa-tRNA on A site
- EF-Tu is a GTP protein and takes the elongator aa-tRNA and places it at A site. When it is put at the A site, GTP is hydrolyzed and becomes GDP. The protein is now inactive
- EF-Tu-GDP then binds to EF-Ts and displaces GDP
- EF-Tu-Ts binds to GTP and displaces Ts
Formation of peptide bond
Done by peptidyl transferase. Located on 23S in prokaryotes and 28S in eukaryotes. Both are located in the large subunit
Transferring from A to P
Use EFG (translocase) to move it from A to P.
Some antibiotics inhibit translocation which terminates translation
Antibiotics that bind to A site inhibit translation
Streptomycin
Binds to 16S on small subunit and prevents binding of initiator aa-tRNA to P site. Leads to polypeptide frame shift. Inhibition in initiation
Termination
Stop codons UAA, UAG, and UGA don’t have any tRNA association with them
- RF 1/2 will releases the polypeptide
- RF3 dissociates it and proofreads it
If a tRNA is put at a stop codon
-RF3 terminates it and RF1/2 releases it
This is post quality control
Streptomycin
Misreading of mRNA
Tetracycline
Inhibits elongation
Chloramphenicol (pro) or cycloheximide (euk)
Inhibits elongation
Erythromycin
Inhibits elongation
Duromycin
Inhibits elongation
Diphtheria toxin
Inhibits translocation
Protein reaches its target by…
- Targeting sequence
- Specific modification (protein folding, glycosylation)
Protein modification
- Cleavage: make inactive proteins (zymogens) active. Ex: digestive enzymes, preprohormones (insulin)
- Hydroxylation: collagen
- Phosphorylation: TFs
- Lipidation: adding lipid anchors
- Glycosylation: membrane proteins and secretory proteins
Category 1 protein
Synthesizes entirely on the free ribosomes
Include: mitochondria, nucleus, proteosomes, and cytoplasm proteins
Category 2 proteins
Begins synthesis in the free ribosome and then completes it on the rER.
Includes: plasma proteins, ER and Golgi resident proteins, lysosomal proteins, and secretory proteins
Category 3 proteins
Synesthesia complete on the free ribosome but is then moved to the ER for post-translational modification
Mitochondrial proteins
Have a target sequence that is on the N-terminus. Is cleaved when the protein reaches the mitochondria
Nuclear proteins
Has their target sequence on the N-terminus. Is not cleaved when it reaches the nucleus
Proteosomal proteins
Hydrolyze fat. Have their target sequence on their C-terminus attached to Ser-Lys-Phenalalanine
Coupling mechanism
Signal peptide (or leading sequence) is located on N-terminus and this will be recognized by signal receptor particles (SRP). SP-SRP complex will slow down translation and bind to SRP receptor on rough ER. Once it binds, GTP will be hydrolzed. Translation will then resume
Sometimes, the signal peptide is cleaved by the ER
Modifications in the ER
- Glycosylation
- Oligomerization
- Quality control
- Protein folding
- Forming disulfide bridges
Glycosylation
N- linked Glycosylation: attaching complex carbs on the N terminus to Asn
O-linked glycosylation: attaching complex carbs on OH to Ser/Thr
Protein folding
Chaperones bind to the exposed hydrophobic amino acids to help fold properly
Quality control
If a protein is not folded properly, glucosyl tranferase will add glucosyl tranferase enzyme. A chaperone called calnexin will fold the protein correctly.
If the protein still isn’t folded correctly, will go to peroxisome.
If the protein is folded correctly, G molecule will be removed by glucosidase II and will go to Golgi
Modifications on the Golgi
- Terminal glycosylation
- O-linked glycosylation
- Sorting/packing
Sorting lysosomal proteins
Lysosomal proteins are tagged with mannose-6 phosphate and its receptor. It is exocytosed to an endosymbiosis which recycles the mannose-6 phosphate and the receptors
ER resident proteins
Have KDEL. When it goes to Golgi, it will have KDEL receptor to send the protein back to the ER
Mucolipidosis II
Accumulation of inactive lysosome and so accumulation of mucopolysaccharides in inclusion bodies (mostly in fibroblasts)
4 levels of protein structure
- Primary
- Secondary
- Tertiary
- Quaternary
Primary structure
Linear structure of amino acids that is stabilized by peptide bonds (covalent bonds)
Secondary structure
Stabilized by H bonds between the amino and carboxyl group.
5 forms:
- a helices
- B sheets
- B turns
- Repetitive sequences
- Motifs
A helices
In a right handed helix. Stabilized by H bonds between the 1st and 4th amino acid respectively.
Also have:
- a 10 helix: between every third amino acid
- Pi helix
B sheets
Formed by two segments of a polypeptide. If it’s formed by two polypeptides, leads to disease. R groups are up or down.
Have two sheets:
- Antiparallel: segments are coming opposite ways. Major form
- Parallel: segments at coming from the same way. Minor form
B turns
Abrupt changes in B sheets. Glycine and proline help stabilize it. Four amino acids make up the turn, of which glycine and proline are present. Bond between 1st and 4th amino acid and 3rd and 4th amino acid
Non-repetitive sequences
Loops and coils
Super secondary structure (motifs)
2 or more secondary structures that are connected by loops or turns
- Helix-turn-helix: major groove binding
- Helix-bend-helix: TFs
- B barrels: antiparallel rolled up (bacterial pores)
- B hairpins: found in globular proteins
- Helix bundles: transmembrane proteins
Tertiary structure
Stabilized by ionic bonds, H bonds, hydrophobic interactions, and disulfide bridges
Quaternary structure
2 or more polypeptides
Protein denaturation
Disrupts the non-covalent interactions but not the covalent bonds (peptide and disulfide)
Denaturants: pH, heat, urea, organic solvents
Enzymes
Lower activation energy but don’t change delta G
Standard unit
1micromol/min.
This is how you measure enzyme activity since you can’t get molar amount of enzyme
Specific activity
IU/mg
Measure enzyme purity
Isoenzymes
Enzymes that catalyze the same reaction in different parts of the body
CK2
Isoenzyme found in cardiac tissue. Is a diagnosis marker for cardiac tissue damage since damage leads to a lot of CK2 release
Cofactors
Other substances needed to help in enzymatic activity
2 types:
- Inorganic
- Organic: coenzymes
Ribozymes
Non-protein enzymes that are made of RNA. Help catalyze peptide bond formation
Ribonuclearproteins
Made of RNA and nucleic acids.
Ex: telomerase
Proximity effect
Brings enzyme and substrates together
Orientation effect
Provides correct orientation for catalysis
Catalytic effect
Provides the correct functional groups (acidic, basic)
Energy effect
Helps reach transition state
Lock and key model
Substance fits directly into the enzyme.
Static model
Induced fit
Enzyme wraps around substance since they are not a perfect fit
Dynamic model
V max
Maximum rate that is achieved theoretically
Km
Substance concentration that reached 1/2 v max
Reversible inhibitors
Bind to enzyme through non-covalent interactions. If you add a diluted solution without inhibitors, inhibitors will be washed off
Irreversible inhibitors
Covalently binds to the enzyme. Adding the solution won’t wash off the inhibitors
Ex: lead and mercury bind to S on cysteine residues
Ex: malathion (organophosphorus insecticide) and sarin (nerve gas) inhibit acetylcholinesterase
Competitive inhibitors
Actively competes with the substrate to get to the active site. Higher substrate concentration will displace inhibitor. So max isn’t changed by km is increased
Lovastatin
Lovastatin inhibits HMG-CoA reductase (synthesizes cholesterols) to reduce cholesterol levels
Good for treatment of hypercholesterolemia
Non-competitive inhibitors
Binds to the enzyme at a different site than the active site. Leads to change in active site or getting rid of it altogether. Decereases vmax but km is the same
Transition state analogs
Mimics the 3D structure of the transition state but doesn’t form products. Bind very tightly to the active site
Suicide inhibitors
Binds to the enzyme but never leaves the active site
Ex: penicillin inhibits glycopeptice transpeptidase by modifying the serine chain -OH group
Ex: aspirin inhibits cyclooxygenase
Ex: allopurional inhibits xanthine oxidase
Changing enzyme amount
- Synthesis: slow
- Proteolysis: fast and degrades
Changing enzyme activity
- Proteloysis
- Phosphorylation/dephosphorylation
- Cooperativity/alosteric regulation
Trypsinogen
In its inactive form. Be woke active by cleavage (proteolysis) or 6 amino acids. Active ones can then activate other inactive ones
Cooperativity
Usually when an enzyme has more than one subunit. One substrate binds to active site on one of the subunits and if it’s positive cooperativity rest of the substrates will bind as well.
Does not follow Michaelis-Mentin curve. Leads to a sigmoid also curve
Alosteric regulation
Effector binds to place outside of the active site and can be positive or negative effect.
When paired with Cooperativity, activators will stabilize the high energy state and make the curve more hyperbolic
V effector
Affects vmax parameter and is used when enzyme has higher enzyme concentration
Km effector
Affects km parameter and is used when enzyme has low substrate concentration
Adding phosphate by…
Protein kinase with the help of ATP
Taking away phosphate…
Phosphoprotein phosphatase
Protein kinase A
Ligand binds to membrane which sends secondary messenger (cAMP) which tells kinase to phosphorylate and become active. Kinase then works on multiple reaction pathways
Glycogen phosphorylase
When phosphorylated, increases activity
Glycogen synthase
When phosphorylated, decreases activity
Preparing agarose gel
- One gram of agarose in 50 ml of TBE buffer
- Microwave for 1-2 mins
- Put under running water
- Put in casting tray its comb and make sure there are no air bubbles
- Let it solidify
- Take out comb
PCR method
- Add MgCl2, taq polymerase, dNTP into lambda DNA and primers
- Centrifuge
- Add buffer dye (helps see DNA migration)
MgCL2
Helps in primer binding to DNA
Electrophoresis method
- Take 10 micro liters or marker and put in one well
- Put 20 microliters of sample in one well
- Run for 1 hr at 60 volts
- Look at results with UV trasilluminator camera
Sickle cell anemia
Point mutation in changing glutamate to valine. This makes it stay at the top of gel electrophoresis.
Mutation in B chain of hemoglobin
Normal:
- Oxygenated: nothin
- Deoxygenated: pockets
Mutated:
- oxygenatedm spikes
- Deoxygenated: spikes and pockets ( so forms clumps when low oxygen which leads to vessel clogging and leads to pain)
Prions
Change alpha helices to b sheets. Leads to rogue protein which can make enormous protein rogue as well
Exam mad cow disease, CFJ
Amyloid precursor protein
Not cleaved properly so leads to Alzheimer’s since plaques develop
Collagen synthesis
In the rough ER. Formed by C terminus and helped by inter and intra H bonds as well as disulfide bridges
In the extracellular space, N and C peptodases will cut off some of the N and C ends
Modification of collagen
- Hydroxylation of proline by proloyl hydroxylase with the help of vitamin C
- Fibril formation by covalent cross-linking of lysine helped by lysine oxidase. Need copper for this
OI
Change in glycine to a bulky amino acid will result in a weak and brittle collagen and no hydroxylation
EDH
Caused by mutation
UV radiation
Degrade collagen and forms wrinkles and premature aging
Metabolic pathways
All reactions in a cell
Cycles
Pathways that regenerate a substance
Catabolic pathways
Breaking down a high energy product (glucose) to a low energy product (CO2)
Produces ATP, NADH, and NADPH
High ATP closes catabolic pathways
Anabolic pathways
Building up high energy products from low energy products
Use ATP, NADH to provide energy for the buildup
High ATP open anabolic pathways
Break reactions into small steps
- Pathways will take place where the enzyme is
- Compartmentalized into organelles
- One product will be reactant of the next
Intrinsic regulation
Based on cells needs. Use ATP, ADP, NADPH, NADP+
Extrinsic regulation
Based on body’s needs. Use hormones
Free energy
Energy available to do work. When it’s negative, it’s spontaneous. At equilibrium, delta G is 0.
Can couple a nonspontaneous reaction to a spontaneous one
Phosphory-transfer potential
Ability to transfer a phosphate group. ATP has can both accept and donate a phosphate group
Redox reactions
Transferring electrons between substances
Redox potential
Wanting to gain electrons
Reducing equivalent
Substance that helps transfer electrons
NADH and NADPH are the come common ones
Committed step
First reaction in a pathway that is irreversible and has only one unique product. Also known as the rate-limiting step. It’s also the slowest step
Feedback inhibition
Product of a reaction will inhibit that same reaction
Advantages:
- Prevent accumulation of toxic waste
- Prevents wastage if energy
Feed forward reaction
A product early on in the reaction will activate an enzyme later on in the pathway
GLUT isoforms
Found on the membrane of cells and allows glucose to enter the cell
Glycolysis
Beginning of respiration if oxygen is available. If in anaerobic conditions, it’s the only way to get ATP
Changing glucose to G6P
Phosphorylate glucose using ATP with a hexokinase
This allows:
- Glucose to be be further metabolized since it was inert
- Prevents glucose from leaving the cell because now it’s an anion
NOT THE COMMITTED STEPPPP
Hexokinase
Found in non-hepatic cells
Has a low km so non-hepatic cells can trap glucose
Low vmax so can’t trap phosphorylated glucose or phosphorylate more glucose than it can use
Glucokinase
Found in the liver
Has high vmax for rapid uptake glucose from bloodstream, gkycogenesis, and prevents hyperglycaemia
Has high km fo rapid synthesis/release of glucose (can leave liver following a big meal), glycogenolysis, and prevents hypoglycemia
Induced by insulin
PFK1
Changes fructose 6 phosphate to fructose 1,6 biphosphate through phosphorylation
Aldose B
Cleaves fructose 1,6 biphosphate to glyceraldehyde 3 phosphate
Glyceraldehyde 3 phosphate dehydrogenase
Changes glyceraldehyde 3 phosphate to 1,3 biphosphoglycerate
This is the first oxidation reduction reaction and produce a NADH
Pyruvate kinase
Changes PEP to pyruvate and gain an ATP through substrate level phosphorylation
Branching of pyruvate
- Oxidative decarboxylation to acetyl CoA
- Reduction to lactate and ethanol
- Transformation to alanine
Regulation of hexokinase
Inhibited by G6P. So make sure that if there is a lot of glycolysis going on (meaning there will be a lot of energy) won’t waste any more glucose
Regulation of PFK1
Negative: ATP, citrate, H+ from lactate
Positive: fructose 2,6, biphosphate and AMP
Fructose 2,6, biphosphate
Most important regulator for PFK1. Not an intermediate of glycolysis. Catalysts by PFK-2
PFK2
Non-phosphorylation form catalyze fructose 2,6 biphosphate
Phosphorylated form: glucagon will bind to membrane causing cclyic aMP to activate protein kinase A which will phosphorylate PFK2. Liver stops consuming glucose and makes glucose (so opposite effect!)
In the heart, the phosphorylated form is active and vice versa
Regulation by pyruvate kinase
Negative: alanine, ATP
Positive: fructose 1,6 biphosphate: feed forward reaction
Fructose accumulation
When there’s an aldose B deficiency, accumulation of fructose and deficiency in ATP
Shouldn’t give people fructose or sucrose
Classic galactosemia
Deficiency of GLAT which changes galactose you glucose so accumulation of galactose and can’t make ATP
Get acetyl coA from…
- Pyruvate
- Fatty acids
- Proteins
Pyruvate dehydrogenase
Changes pyruvate to acetyl CoA
Need it to be inactivated so don’t need the protein kinase A
Positive: ATP, NADH
Negative: ADP, NAD+, CoA
Oxoacetate
Transports CoA to cell
Citrate
Can move in the mitochondria
ACETYL COA CANT MOVE INSIDE THE MITOCHONDRIA
Isocitrate dehydrogenase
Rate-limiting step
Negative: ATP and NADH
Positive: ADP, Ca
Uses both NAD and NADP
Succinyl coA
Metabolic branch point
Succinyl thiokinase
Makes the GTP
Succinate dehydrogenase
Complex II in mitochondria electron transport chain. Changes FAD to FADH2
Malaga dehydrogenase
Generates last NADH
Summary of Krebs cycle
- 2 carbons from acetyl CoA released as CO2
- Make 3 NADH
- 1 FADH2
- 1GTP
Regulation of Krebs cycle
- Citrate synthase inhibited by citrate
- Allosteric regulators of cycle: Ca2+, ATP, ADP
Inner membrane of mitochondria
Not permeable to substances. Has five complexes
Four are for transferring electrons
One is for making ATP
Inner membrane is folded to allow the complexes to be on it
Intermembrane space
Has a lot of H+ ions
Matrix
Place where the hydrogen ions go
Electron transport chain
Electrons lost by NADH & FADH2 and go to oxygen and combine with hydrogen to form water
Oxidative phosphorylation
Using diffusion energy of H+ ions, can make ATP
Complex 1
NADH dehydrogenase. Has Fe-S centers, FMN, and coenzyme Q
Fe-S will transfer electrons from NADH to coenzyme Q
Allows proteins to go into the intermembrane space
Coenzyme Q (ubiquinone)
A benzoquinone which is hydrophobic.
Gets one electron to be a emiubiquinone radical and two to become ubiquinone
Small size allows for diffusion across the membrane.
Statin inhibits it’s synthesis
Flavoproteins
Contain FMN or FAD.
Accept one electron to become semiquinone and 2 to become FMNH2
Can donate two electrons to NAD+ and one to Fe
Fe-S
Contain 2-8 iron atoms that are complexes with elemental and cysteine S residues
Can only transfer one electron at a time
Complex 2
Succunate dehydrogenase (like in TCA). Has FAD and Fe-S clusters
Electrons from Coq are given to FAD which reduce it. FAD gives to Fe-S clusters who give it to another Coq
Complex 3
Cytochrome c reductase. Contains cytochrome c, b1, Coq.
Undergoes the Q cycle
Q cycle
Round 1: CoQ gives electron to Fe-S who gives it to to cytochrome c. Other electron goes to cytochrome b. At this time, CoQ will dock at cytochrome b point and receive this electron to become a radical
Round 2: Another CoQ will come and give electron to cytochrome c. Other will go to cytochrome b to give to quinone radical to become normal again
Cytochromes
Contain heme which can undergo transition between two iron states
Cytochrome c
Transfers electrons to complex 4. Has positive amino acids which interact with the negative amino acids in complex 4 which helps it dock
Complex 4
Cytochrome C will dock on complex 4 and Cu will accepts the electrons and give it to O one at a time
1 e: o radical
2 e: peroxide
3 e: 2 o radical
4 e: water
Moving H
Complexes will have strong affinity for H in the beginning and then have low affinity which will allow it to release it
Complex 5
Makes ATP
Has two parts: F1 and F0
F0
Has a rod (a subunit) and rotating part (c subunit). Protons go up rod and are repelled by c subunit. End of c subunit when bound to rod will allows hydrogen ions to exit leading to a step movement
F1
In the matrix and makes ATP
Has 3 catalytic sites
Site 1: has ADP and pi
Site 2: has ATP
Site 3: empty
Every rotation is 120 degrees to repeat the cycle after every three rotations
Uncouplers
Lipid-soluble molecules that will dissipate the electrochemical gradient of H+. H ions will still move but instead of energy being in ATP, it’ll be in heat
Thermogenein (UCP1) is an example
UCP2 and UCP3 lead to weight loss by breaking down fat
Mitochondrial diseases
From mother. Random selection of mitochondria region leads to some areas having more infectious agents than others
Glycogen
Stores glucose. Is a branches polysaccharide that is sparsely soluble
Has glycosidic type a 1,4 and 1,6 bonds
Found in liver and muscle. Higher composition in liver but higher mass in muscles.
Muscle glycogen can not give glucose to other tissues since it doesn’t have glucose 6 phosphorylase
Phosphoglucomuatse
Isomerizes glucose 6 phosphate to glucose 1 phosphate
Glycogen synthase
Adds glucose to 4’ end on glycogen branch. However, this is thermodynamically unfavorable so first has reaction with UTP and glycogen synthase uses this phosphate to add glucose
Glycogen synthase can only add glucose to an already existing glycogen chain
If no glycogen chain…
Glucose attaches to tyrosine residue on glycogenin. Glycogenin catalyzes it’s own reaction
After about 7 chains, glycogen synthase can begin acting on it
Branching enzyme
Takes a residue of about 6-7 branches and attaches it to a terminal carbon 6 group to form an a 1,6 bond
Glycogen phosphorylase
Breaks down glycogen to form glucose. Cannot break down a glycogen chain that is 4 residues or less
Debranching enzyme
Breaks down the 4 chain glycogen molecule. Takes three out of the four glucose molecules and adds them to a longer chain. Takes the remaining one and cuts off the bond to form a free glucose molecule
Lysosomal degradation of glucose
Lysosome has enzymes to break down glycogen. However, breaks down a very negligible amount.
When this enzyme is defected, glycogen accumulates in the lysosome vesicle and leads to glycogen storage disease type II (pompe’s disease)
Regulation of glycogen phosphorylase
When blood glucose becomes low, glucagon levels become high. So glucagon binds to the receptor and form cAMP. cAMP will activate protein kinase A. PKA will activate the active “a” form of phosphorylase kinase. “A” form of phosphorylase kinase will activate glycogen phosphorylase and break down glycogen
In muscle, epinephrine is used
A adrenergic receptors
Couple to adenyl cyclase and form cAMP
B adrenergic receptors
Coupled to G protein. G protein activates phospholipase C. Phospholipase C hydrolyzes PIP2 to form IP3 and DAG
DAG binds to & activates protein kinase C that phosphorylates a lot of molecules including glycogen synthase
IP3 binds to ER receptors to release Ca. Ca will bind with calmodulin and activate phosphorylase kinase
In liver, epinephrine…
Leads to same Ca cascade
In muscle, acetylcholine
Will release Ca and go through same cascade. In this way, don’t need pka to activate phosphorylase kinase
AMP also activated phosphorylase kinase
Glycogen synthase
Exits in active “a” non-phosphorylated form and inactive “b” phosphorylated form
Insulin will activate glycogen synthase and glucagon will inhibit it
Pentose phosphate pathway
Anabolic reaction that changes 6 carbon sugar (glucose) to 5 carbon sugar (ribose) for nucleotide and nucleic acid synthesis
Also makes NADPH and makes glyceraldehyde-3-phosphate and glucose-6 phosphate
Has 2 steps:
- Oxidative
- Non oxidative
Oxidative
G6P is changed to 6-phosphogluconate and produces NADPH
6-phosphogluconate is changed to ribulose-5 phosphate and produces NADPH
NADPH
Differs from NADH by one phosphate group. Involved in anabolic reactions
Need it for:
- Synthesis of fatty acids and cholesterol
- Changing ribonucleotides to deoxyribonucleotides
- Reducing glutathione (antioxidant)
Non-oxidative stage
- Change ribulose 5P to ribose 5P (make NADPH)
- Make glyceraldehyde 3-phosphate and G6P (to go back to oxidative stage and g9 to glycolysis) (also makes NADPH)
Digesting proteins in stomach
- HCl: secreted by parietal cells and first denatures and then cleaves
- Pepsinogen: gets activated by active pepsin and HCl
Digestion by pancreas
Zymogens released by cholecystokinin and secretin
Activated by:
- Enteropeptidase activates trypsinogen
- Trpsinogen activates everything else
Digestion in small intestine
Done by aminopeptidase which is an exopeptidase the is found in the lumenal surface of the intestine. Repeatedly cleaves N-terminus from oligopeptides to form smaller peptides and amino acids
Absorbing free amino acids
Through Na+ linked secondary transport system in RBCs
Absorbing di- and tri- peptides
Through a H+ transport system
Cystinuria
Defect in transport system in proximal tubules of kidney to absorb cysteine. Leads to accumulation of cysteine leading to stones in the urinary tract
