Final extras Flashcards
1
Q
What is a exo- and endoprotease?
A
Exoproteases cleave a protein at its ends; aminopeptidase and carboxypeptidase
Endoprotease cleace within a protein
2
Q
Which two amino acids has two chiral chains?
A
Threonine and isoleucine
3
Q
Which AA has a charges sidechain at pI?
A
Argenine and lysine
4
Q
What are characteristic for alpha chains?
A
Maximum amount of H-bonds. 3.6 residues per turn. Most often right handed. Primary structure in alpha-keratin. H-bond between residue 1 and 4. Sidechain are pointet out.
Glycine and proline decreases its stability (there are many other factors..)
5
Q
What are characteristic for beta sheets?
A
H-bonds are either in line parallel or anti-parallel.
6
Q
What are characteristic for beta-turns?
A
Reverse the direction of the polypeptide chain. 4 amino acids. 1st AA carbonyl oxygen forms H-bond with 4 AA amino group.
Proline and glycine often occur in B-turns.
7
Q
Which interactions are important for a proteins tertiary structure?
A
Disulfide bonds Hydrophobic AA at the interior H-bonds between polar AA Ionic interactions Hydrophilic surface
8
Q
What is the normal AA code in collagen?
A
Gly-X-Y, where X and Y is proline and hydroxyproline (or hydroxylysine)
9
Q
Keratin consists of how many strands?
A
Two. Create a left-handed helix. Rich in hydrophobic AA (not proline)
10
Q
List 5 general facts about enzymes
A
Activation energy decreases Delta G does not change The reaction mechanism change Eq state does not change Time to reach EQ is decreased
11
Q
A nucleotid are made up by? What are the name of the bond between two nucleotids?
A
a nitrogenous base, a five-carbon sugar (ribose or deoxyribose), and one phosphate group.
3´5´ phosphodiesterbond (3carbon on one ribose to a 5 carbon on the other ribose)
12
Q
What bases do we have?
A
Purine: Guanine, adenine
Pyramidines: Thymine, cytosine
13
Q
Adenine and thyme + Cytosine and Guanine has how many H-bond
A
A-T = 2 C-G = 3
14
Q
RNA when formed is first “protected” by modification of the 3 and 5 end by what?
A
3 end = poly adenine groups. 200+
5 end = Cap (methylated guanine triphosphate)
15
Q
What is typical about Scurvy?
A
Scurvy is a disease resulting from a deficiency of vitamin C. Humans and certain other animal species require vitamin C in their diets for the synthesis of collagen
16
Q
Replication of DNA occur in which stage of the cell cycle?
A
S-phase
17
Q
Describe these stages, G1, S, G2, M in the cell cycle
A
G1 = prepare to duplicate S = synthesis of DNS G2 = prepare for cell division M = cell division
G0 = “cell rest”
18
Q
DNA polymerase read and make nucleotides in what direction?
A
Read: 3-5
Make DNA: 5´-3´
19
Q
What is the name of the antibiotic that work on prokaryotic RNA synthesis?
A
Rifampicin
20
Q
Anomers
A
Isomeric form of monosaccarides that differ in the conformation around the hemiacetal or hemiketal carbon bond, known as a anomeric carbon
alpha and beta conformation
21
Q
Epimer
A
Two sugars that differ only around one carbon.
E.g. Glucose and mannose is C4 epimers
22
Q
What is the bond name between two monosaccarides? What monosaccarides is present in maltose, lactose and sucrose?
A
Glycosidic bond. 1-4 linkage
Maltose = 2x glucose - alpha 1-4 Lactose = Glucose + galactose - beta 1-4 Sucrose = Glucose + fructose - beta 2 - alpha 1
23
Q
What is the difference between amylose and amylopectin? That is special about cellulose?
A
Amylose has only alpha 1-4 bonds
Amylopectin is “plant glycogen”. Has 1-6 branching points
Cellulose can not be broken down in the GI- tract. We dont have enzymes that can act on beta 1-4 bonds.
24
Q
What is the function of telomerase?
A
Elongate the ends of DNA strands (telomer). E.g. after DNA replication
25
Histones and its modifications
Octamers (4 dimers). H1 linkage. Positive charged (arginine + lysine). Makes DNA more condense. Nucleosome -> Nucleofilaments.
HAT (histone acetyl transferase) decondese
HDAC (histone deacetylase) condense
26
Topoisomerase function and types
Topoisomerases are enzymes that regulate the overwinding or underwinding of DNA. The winding problem of DNA arises due to the intertwined nature of its double-helical structure. During DNA replication and transcription, DNA becomes overwound ahead of a replication fork. If left unabated, this torsion would eventually stop the ability of RNA & DNA polymerase involved in these processes to continue down the DNA strand.
E.g. Helicase will cause a twisting motion in the DNA strands.
Topoisomerase1: Break a phosphodiesterbond by nucleases without the use of ATP. Link it togheter later by ligases. Have a tyrosine in its active center. Reversable reaction.
Topoisomerase2: ATP-dependent. Break DNA on both strands
27
Name some common features for DNA polymerase
1: 5´-3´polymerase activity (read the strand 3`-5`direction)
2: Need primer to start (free 3´end hydroxyl)
3: Uses DNA a a template
4: dATP, dTTP, dCTP, dGTP
5: PPi product (pyrophosphate)
6: All DNA polymerase have 3`-5`exonuclease activity (proofreading). Type 1 have in addition, 5`-3` exonuclease (removal of RNA primer)
28
DNA ligase function and requirements
The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide, ("acceptor") with the 5' phosphate end of another ("donor"). ATP is required for the ligase reaction, which proceeds in three steps:
1: Adenylation (addition of AMP) of a lysine residue in the active center of the enzyme, pyrophosphate is released;
2: Transfer of the AMP to the 5' phosphate of the so-called donor, formation of a pyrophosphate bond;
3: Formation of a phosphodiester bond between the 5' phosphate of the donor and the 3' hydroxyl of the acceptor. 3` attack this bond. AMP is a good leaving group.
Extra: Has lysine in its active center
29
What is a silent mutation?
Mutation in nonessential DNA
30
Cytosine can undergo spontaneous deamination to?
Uracil
This can make a mutation from C-G to A-T
31
Adenine and guanine can undergo deamination to?
Hypoxanthine and xantine, respectively
32
Nonsense mutation
Stop signal. AA sequence become shorter
33
Missense mutation
Code for different AA. Different protein is formed. Can be conservative or non-conservative
34
Frameshift mutation
Mutation caused by insertion or deletion of nucleotids. This shifts the triplet codon and alters many AA. Can make the protein longer or shorters
35
Epigenetics
Methylation on cytosine nucleotids. Shut off genes
36
Fatty acid metabolism: Lipoprotein lipase is stimulated by?
Insulin and Apoprotein C2 (which is transfered from HDL to VLDL and chylomicrons).
LPL break down TG to FA and glycerol
37
Does adipose tissue have glycerol kinase?
No. They need glucose (stimulated by insulin, GLUT4) to make glycerol 3-P.
38
What is the main difference between HMG-CoA in mitochondria and cytosol?
Cytosol: Cholesterol synthesis
Mitochondria: Ketone bodies synthesis
39
What is the first step in bile acid synthesis (which is also the rate-limiting)?
7a-hydroxylase
Inhibited by bile acids
40
HDL can give which lipoproteins to VLDL and chylomicrons?
ApoC2, ApoE
41
Cholesterol is converted to cholesterol ester by?
Lecithin:Cholesterol acyltransferase (LCAT) - In HDL?
Acyl:Cholesterol acyltransferase (ACAT) - For storage in cells
42
Function fo CEPT (cholesterol ester transfer protein)?
Exchange cholesterol ester with triglycerides. HDL can becomme TG rich
43
What is the difference between primary and secondary carnitine deficiency?
Primary: Inability to transport carnitine into the cells
Secondary: Caused by other metabolic disorders such as CAT2 Mutation, or Fatty acid oxidation disorders.
44
5´cap on mRNA consists of?
7-methylguanosine attached via a triphosphate
45
What makes up prokaryotic RNA polymerase?
2alpha, Beta for 5`-3´polyemerase, Beta` for template binding.
Togheter with the sigma factor (for the recognizion of the promoter) forms a holoenzyme
46
Consensus regions in prokaryotes
-35 and -10 (Pribnow box)
47
P-dependent and P-independent termination of RNA synthesis; what is the difference?
P-independent: RNA is self-complementary. RNA folds back on itsself forming a G-C righ stem plus a loop (hairpin). This facilitate the seoaration from the DNA-RNA hybrid helix. Palindromic , inverse repeat forms a hairpin loop and is believed to physically destabilize the DNA - RNA hybrid.
P-dependent: Require an additional protein, rho, which is a hexameric adenosine triphosphatase with helixase activity. Rho binds to a C-rich "rho recognition site" near the 5-end of the RNA strand until it reach the RNA polymerase paused at the termination site. The ATP dependent helixase separates the RNA-DNA hybrid helix, and release the RNA strand.
48
Consensus regions in eukaryotes
-25 (TATA or Hogness box), -80 (CAAT box)
49
TFIID recognize the promoter region
TFIIF binds the polymerase to to promoter
TFIIH has helixase activity
FU
50
What is splicing?
Removal of introns from exons. Occur in a spliceosome.
small nuclear RNA or snRNPs (snurps) are involved. U1-U6. snRNAs U1, U2, U4, U5 and U6 form complexes with 6-10 proteins each, forming small nuclear ribonucleoprotein particles (snRNPs).
After the formation of the full spliceosome, the U1 and the U4 snRNPs are detached and the remaining U2, U5 and U6 snRNAs are rearranged. This conformational change creates the catalytic spliceosome (transesterfication).
After the second transesterification reaction, the spliceosome comes apart. The snRNPs are recycled, and the spliced exons and the lariat intron are released.
51
What is the name of the enzyme which charge tRNA?
Aminoacyl-tRNA synthetase. Makes aminoacyl-tRNA. Need ATP -> AMP + PPi.
Two step reaction: Covalent attachment of a carboxyl of a amino acid to the 3-end of the tRNA.
Makes a 5`-aminoacyl adenylate intermediate
Aminoacyl-tRNA synthetase has proofreading
aa + tRNA + ATP -> Aminoacyl-tRNA + AMP + 2 Pi
52
Eukaryotic and prokaryotic ribosomes consists of two units. Which one?
Prokaryotic: 50S + 30S -> 70S
Eukaryotic: 60S + 40S -> 80S
53
Initiating of translation. Codon sequence and names
AUG sequence. AUG have N-formylated methione on bacteria and mitochondria. In eukaryotes, AUG code for methionine.
GUG codon can also initiate
54
SRP (signal recognition particle)
The signal recognition particle (SRP) is an abundant, cytosolic, universally conserved ribonucleoprotein (protein-RNA complex) that recognizes and targets specific proteins to the endoplasmic reticulum in eukaryotes and the plasma membrane in prokaryotes..
Its an ribozyme (RNA with enzymatic activity) and GTPase. (GTP for its dissociation). An RNAP complex containig an ancient RNA and six proteins RNA Uses GTP for its function
55
Which two AA is the only one coded by only one codon?
Methionine and tryphophan
56
What is the universal stop codons?
UAA, UAG, and UGA are stop codons and specify
| the the end of translation of a polypeptide.
57
What is "Wobble base pair"?
A wobble base pair is a pairing between two nucleotides in RNA molecules that does not follow Watson-Crick base pair rules. The four main wobble base pairs are guanine-uracil (G-U), hypoxanthine-uracil (I-U), hypoxanthine-adenine (I-A), and hypoxanthine-cytosine (I-C)
58
Name of enzyme that transfer one AA to the growing polypeptide chain in ribosomes
Peptidyl transferase is an aminoacyltransferase
59
What is the Shine-Delgarno site?
The Shine-Dalgarno (SD) sequence is a ribosomal binding site in prokaryotic messenger RNA, generally located around 8 bases upstream of the start codon AUG. The RNA sequence helps recruit the ribosome to the messenger RNA (mRNA) to initiate protein synthesis by aligning the ribosome with the start codon.
60
Which Codons signals termination and how is it done?
Proteins known as "release factors" recognize the stop
codon (UGA, UAG, or UAA) at the A site. In E. coli RF-1 recognizes UAA and UAG, RF-2 recognizes UAA and UGA.
RF-3 binds GTP and enhances activities of RF-1 and –2.
Presence of release factors with a nonsense codon at A
site transforms the peptidyl transferase into a hydrolase,
which cleaves the peptidyl chain from the tRNA carrier.
Hydrolysis of GTP is required for disassociation of
RFs, ribosome subunit and new peptide.
61
Function of eIF2
eIF2 supplies Met-tRNA to 40S subunit. eIF2 phosphorylation inhibits initiation
62
Function of signal peptidases?
Signal peptidases are enzymes that convert secretory and some membrane proteins to their mature forms by cleaving their signal peptides from their N-terminals.
All signal peptidases described so far are serine proteases. The active site that endoproteolytically cleaves signal peptides from translocated precursor proteins is located at the extracytoplasmic site of the membrane. The eukaryotic signal peptidase is an integral membrane protein complex
63
Is the antisense or sense strand used for transcription?
Antisense (template)
Sense strand is the non-template
The direction of transcription on the different strands is opposite. This feature is referred to as asymmetric transcription.
64
Function of RNA polymerase
```
RNA polymerase catalyzes the formation of phosphodiesterase bonds
between nucleotides (using triphosphate nucleotides)
```
65
Function of sigma factor
A sigma factor (σ factor) is a protein needed only for initiation of RNA synthesis. It is a bacterial transcription initiation factor that enables specific binding of RNA polymerase to gene promoters. As RNA polymerase elongate sigma factor looses its association with RNA polymerase. After 10-15 bp.
66
Name one inhibitor of RNA polymerase
1: α-Amanitin is an inhibitor of RNA polymerase II
2: Rifampicin: Important antibiotic. Rifampicin inhibits bacterial DNA-dependent RNA synthesis by inhibiting bacterial DNA-dependent RNA polymerase
3: actinomycin D. actinomycin D is shown to have the ability to inhibit transcription. Actinomycin D does this by binding DNA at the transcription initiation complex and preventing elongation of RNA chain by RNA polymerase
67
Function of cap binding proteins
Nuclear cap-binding protein complex is a RNA-binding protein which binds to the 5' cap of pre-mRNA. The cap and nuclear cap-binding protein have many function in mRNA biogenesis including splicing, 3'-end formation by stabilizing the interaction of the 3'-end processing machinery, nuclear export and protection of the transcripts from nuclease degradation. When RNA is exported to the cytoplasm the nuclear cap-binding protein complex is replaced by cytoplasmic cap binding complex.
68
What is an lariat structure?
a ring of intron segments that has been spliced out of a messenger ribonucleic acid molecule by enzymes. Some introns form a long tail attached to the ring, giving the structure the appearance of a microscopic cowboy lariat.
69
What is the P-O ratio?
Definition: P/O ratio of oxidative phosphorylation
is the ATP produced per oxygen atom reduced by
the respiratory chain. 2,7 for NADH and 1,6 for FADH2.
Explanation: In mito chondria, for each two electrons transferred to ox ygen from NADH or FADH 2 , ten or six protons, respectively, are pum ped out of the matrix . The combination of the elec trogenic exchange of internal ATP for an external ADP by the ADP/ATP translocase and the non-elect rogenic symport of phosphate and a proton by the phosphate carr ier protein adds one proton to the total requir ed to provide ATP to the cellular cytoplasm, and so the bioenerget ic cost to the vertebrate mitochondrion is 3.7 protons . Therefore, the number of moles of ADP phosphoryl ated to ATP per two electrons transferred to oxygen, (P/O ratio), will be 10/3.7 and 6/3.7, or 2.7 and 1. 6 for NADH and FADH 2 , respectively.
70
Name one drug that inhibits electron transport chain
Oligomycin. Oligomycin A is an inhibitor of ATP synthase. In oxidative phosphorylation research, it is used to prevent state 3 (phosphorylating) respiration. Oligomycin A inhibits ATP synthase by blocking its proton channel (Fo subunit), which is necessary for oxidative phosphorylation of ADP to ATP (energy production)
71
Malonate is an competitive inhibitor of?
Succinate Dehydrogenase
72
Cyanide inhibits?
Complex 4 (Cytochrome c oxidase)
73
Lipid rafts consists of?
Cholesterol- and sphingolipid-enriched, highly dynamic, submicroscopic (25–100 nm diameter) assemblies, which float in the liquiddisordered lipid bilayer in cell membranes.
74
Function of DGAT (Diglyceride acyltransferase)
DGAT, catalyzes the formation of triglycerides from diacylglycerol and Acyl-CoA. The reaction catalyzed by DGAT is considered the terminal and only committed step in triglyceride synthesis and to be essential for the formation of adipose tissue.
Monoacyglycerol acyltransferases (MGATs)
75
Adaptins are important in?
Clathrin formation. Adaptins is the link between the cargo receptors and the clathrin. Formation of a polyhedral lattice
Adaptins are proteins that mediate the formation of vesicles by clathrin-coated pits, through interaction with membrane-bound receptors
Coat disassembly by hsc70 (ATPase)
Dynamin (a GTPase) pull the clathrin coated vesicle of the membrane.
76
Microtubles are made up of many?
Hetrodimers (alpha and beta tubulin). Connected with ATP they from filaments, with ADP they dissociate.
77
Difference between senscent and quiescent cell
```
Senescent cells:
they have stopped dividing
Quiescent cells:
cell cycle is postponed until
an appropriate signal (GF) is received
```
78
What is the steps in base-excision repair?
DNA glycosylase, AP-endonuclease, DNA polymerase 1 or E, DNA ligase
79
Steps in Nucleotide Excision Repair
Nucleotide excision repair (NER) is a particularly important excision mechanism that removes DNA damage induced by ultraviolet light (UV). UV DNA damage results in bulky DNA adducts - these adducts are mostly thymine dimers and 6,4-photoproducts. Recognition of the damage leads to removal of a short single-stranded DNA segment that contains the lesion. The undamaged single-stranded DNA remains and DNA polymerase uses it as a template to synthesize a short complementary sequence. Final ligation to complete NER and form a double stranded DNA is carried out by DNA ligase.
NER use UV-spesific endonucleases. UVRa, UVRb, UVRc and UVRd. Are involved. UVR = Ultraviolet light repair. The process of nucleotide excision repair is controlled in Escherichia coli by the UvrABC endonuclease enzyme complex, which consists of four Uvr proteins: UvrA, UvrB, UvrC, and DNA helicase II (sometimes also known as UvrD in this complex).
Xeroderma pigmentosum
80
In total, how many ATP is needed to add one amino acid to a growing polypeptide chain?
4 ATP/AA
81
Initiation of translation in prokaryotes need?
IF1, IF2, IF3
First 1+3, then 2 later on
IF-3 prevent 50S to bind
IF-2 bind to A-site and prevent other aminoacyl-tRNA to bind in the initiation phase.
82
Initiation of translation in eukaryotes
eIF4 bind to 5-cap of mRNA on 40S. Then, 40S try to find the first AUG sequence on mRNA (ATP-dependent).
Steps:
1: eIF3 + eIF1 bind to 40S.
2: eIF2 binds to P-site with tRNA (has GTP on it), eIF5b-GTP
3: eIF4 with mRNA finds AUG sequence
4: eIF5B hydrolyse eIF2 with GTP
5: initiation factors dissociate
83
Which part of the ribosome have peptidyltransferase activity?
23S
84
The elongation process in ribosomes need
GTP when ribosomes moves to the next codon
85
LAC operon: What is it and what does it code for?
Operon is a group of genes which are regulated and transcripted togheter. One promoter and one operon close to start side. Operon have one repressor. LAC operon contains genes for the transport and metabolism of lactose.
Activators: cAMP (inducer) -> Bind to CAP. Allow RNA polymerase to bind to promoter.
LAC repressor: allolactose
Genes: Lac Z, Lac Y and Lac A -> B-galoctisidase, lactose permease, galactoside O-acetyltransferase
86
What is desmolase and where can we find it?
Desmolase enzyme (20,22-desmolase): Oxidase. Inner MTM. NADPH + O2.
Rate-limiting!
Cholesterol side-chain cleavage enzyme, also called 20,22-desmolase; converts cholesterol to pregnenolone (+ isocaproaldehyde (C6))
87
What is StAr?
Sterogenic regulatory acute protein. Transport protein that regulates cholesterol transfer within the mitochondria, which is the rate-limiting step in the production of steroid hormones. It is primarily present in steroid-producing cells, including theca cells and luteal cells in the ovary, Leydig cells in the testis and cell types in the adrenal cortex.
Activators: cAMP (LH + ACTH). Angiotensin 2.
88
Name of inhibitor for mitotic spindle
colchicine
89
CYP11B1 and CYP11B2 is found where?
CYP11B2 in zona glomerulosa
| CYP11B1 in zone fasiculata and reticularis
90
Aldosterone synthase consists of?
11-Hydroxylase (CYP11B1)
18-Hydroxylase (CYP11B2)
11-Dehydrogenase
Found in the mitochondrium
91
Androgen synthesis require both...
17-hydroxylase and 17,20 lyase
92
Signs of aldosterone excess and deficiency
Excess: Hypertension, hypokalemi, alkolosis
Deficiency: Loss of salt and water, hyperkalemi, acidosis
93
What is the function of 11beta-hydroxylase steroiddehydrogenase 2?
Convert cortisol to cortison (NAD+ dependent).
Cortisol, a glucocorticoid, binds the glucocorticoid receptor. However, because of its molecular similarity to aldosterone it is also capable of binding the mineralcorticoid receptor. Both aldosterone and cortisol have a similar affinity for the mineralocorticoid receptor; however, there is vastly more cortisol in circulation than aldosterone. To prevent over-stimulation of the mineralocorticoid receptor by cortisol, HSD-11β converts the biologically active cortisol to the inactive cortisone, which can no longer bind to the mineralocorticoid receptor. 11β-HSD co-localizes with intracellular adrenal steroid receptors. Licorice or Carbenoxolone, which contains glycyrrhetinic acid, can inhibit 11β-HSD and lead to a mineralocorticoid excess syndrome.
11bOHSDH2: Dehydrogenase - Cortisol inactivation, Cofactor: NAD+,
ER cytosolic surface
Epitelial MR target cells
11bOHSDH1: Reductase- Cortisol activation,
Cofactor: NADPH+H+,
ER luminal surface,
Liver, Adipose tissue, Lung
94
Name of disease and symtoms of excess cortisol
Cushing syndrome
```
Impaired glucose toleranse (DM)
Central obesity
Osteoporosis
Muscle atrophy
Stria
Increased risk of infections
```
95
Name of disease and symtoms of cortisol dificiancy
Adrenal cortex insufficiency
Low BP, dehydration, K+ increase, Na+ decrease, impaired hari growth, exhaustion.
96
What is the problem with "Congenital adrenal hyperplasia"?
Partial or compleate deficiency of 21hydroxylase or 11betaHydroxylasen (CYP11B1)
No cortisol to suppress ACTH secretion: hyperplasia + production of excess androgens, starts in utero
97
ASAT and ALT need which cofactor?
PLP
98
What is the name of the enzyme which remove NH2 group from Glutamate? what does this enzyme need which cofactors? Regulation?
Glutamate dehydrogenase (GLUD). Need NAD+ or NADP+. This is a oxidative deamination reaction.
Activator: ADP + GDP
Inactivator: GTP + ATP + NH4+
GLUD1: Liver, kidney, other tissues
GLUD2: Brain + testies
99
What is the name of the two most common mechanism in humans to transport ammonia from the peripheral tissue to the liver?
Glutamine (by glutamine synthetase. Glutaminase in the liver). Glutamate + ATP + NH3 -> Glutamine + ADP + Pi
Alanine (alanine aminotransferase)
100
What is transdeamination?
Aminotransferase + Glutamate DH reaction
101
Reaction equation for the formation of carbamoyl phosphate. What is the regulations for this enzyme. Where does the second NH3 group come from?
Carbamoyl phosphate synthetase 1. In the mitochondria. Rate-limiting step. Free NH3 is formed by glutamate DH. Regulation: Activators -> N-acetylglutamate (this formation is stimulated by arginine).
NH3 + 2 ATP + CO2 -> Carbamoyl phosphate + 2ADP + Pi
The use of 2 ATP makes this reaction irreversable
Second NH3 from aspartate
Note: Carbamoyl phosphate synthetase 2 participates in the biosynthesis of pyrimidines.
102
List the enzymes in urea formation. How many enzymes do we have? How many ATP are needed per cycle?
1: Carbamoyl phosphate synthetase 1 (2ATP -> 2AMP + 2Pi)
2: Ornithine transcarbamoylase
3: Arginiosuccinate synthetase (ATP -> AMP + PPi)
4: Arginosuccinate lyase
5: Arginase
4 ATP/cycle
The two first enzyme reactions occur in the mitochondria
Overall stoichiometry: Aspartate + CO2 + 3ATP + NH3 + H2O -> Urea + AMP + 2 ADP + 2 Pi + 2 PPi + Fumarate
103
N-Acetylglutamate is formed by? Activator? What does this molecule stimulate
Acetyl-CoA + Glutamate by N-acetylglutamate synthetase
Activator: Arginine
Stimulate Carbamoyl phoshate synthetase 1
104
Asparagine synthetase and glutamine synthetase need?
ATP (Glutamate use 1 phosphate, Aspartate use 2 phosphates -> ATP -> AMP + PPi)
GS need free NH3
AS need glutamine as the NH3 donor
105
Which 7 intermediate does the carbon skeleton of amino acids form?
Pyrovate, Acetyl-CoA, Alpha-ketoglutatare, Oxaloacetate, Succinyl-CoA, Acetoacetate, Fumarate
106
Which Amino acid is the only exclusice ketogenic AA?
Leucine and lysine
107
Which AA form alpha-ketoglutarate via glutamate? 4 AA
Histidine
Proline
Arginine
Glutamine
108
Which AA form pyrovate? 5 AA
```
Alanine
Serine
Threonine
Glycine
Cystine
```
109
Serine can be converted to? 2 pathways
Pyrovate by serine hydratase (Serine -> Pyrovate + H20 + NH4+)
or
Glycine by serine hydroxymethyltransferease (PLP dependent)
(Serine + tetrahydrofolate Glycine + N5-N10 methylenetetrahydrofolate)
110
Which 2 AA can form fumarate?
Phenylalanine and tyrosine
Phenylalanine -> Tyrosine via Phenylalanine hydroxylase in a tetrahydrobiophetin dependent way + O2
They form both fumarate AND acetoacetate
111
Which AA form succinyl-CoA? 4 AA
Methionine, valine, isoleucine, threonine
Valine, isoleucine and threonine (first to alpha-ketobutyrate) -> propinyl CoA -> succinyl CoA. (Threonine can also be converted to pyruvate)
Threonine -> a-ketobutyrate via threonine dehydratase (PLP)
alpha-ketobutyrate -> propionyl CoA -> Methylmalonyl CoA -> Succinyl CoA
1. a-ketobutyrate dehydrogenase complex
2. propionylCoA carboxylase (biotin, ATP)
3. methylmalonylCoA racemase
4. methylmalonylCoA mutase (vitB12)
112
SAM (S-adenosylmethionine) formed from? Remade from? What is the fate of homocysteine
Methionine + ATP via S-Adenosylmethionine + Pi + PPi
Use 3 ATP in total!
Used methyl group -> S-adenosylhomocysteine (SAH) -> hydrolysed to via SAH hydrolase -> Adenosine + homocysteine
Homocysteine can be converted back to methionine or to cysteine.
Homocysteine + N5-Methyltetrahydrofolate -> (Methioine synthetase, need vitB12)) -> Methioine + tetrahydrofolate
Homocysteine + Serine -> (cystathionine synthetase, B6, PLP) -> Cystothionine -> (Cystathionase, B6) -> Cysteine + alpha-ketabutyrate + NH4+
113
What is the deal with "folate trap"?
Vitamin B12 deficiency. Can`t use methionine synthetase, a vitB12 dependent enzyme and use N5-methyl THF. This "trap" THF as N5-THF. Less THF and N5-N10 methylene THF for nucleotide synthesis.
The conversion of 5,10-methylen-THF into 5-methyl-THF, which is catalysed by MTHFR, is irreversible
High adenosine and homoCys in the blood
Methyl trap” (high CH3-H4F, low CH2-H4F)
Inhibition of cell division
114
How do we make N5-methyl THF from THF?
THF + serine -> Via serine hydroxymethyltransferase -> Glycine + 5-10 methylene THF
methylene H4F reductase (MTHFR) use NADH -> 5-methyl THF
N5-N10-methenyl-H4F can be converted to N5-N10 methylene THF via a reduction with NADPH
115
Which two enzymes in the human body need vitB12?
Methionine synthetase (SAM-cycle) and methylmalonyl CoA mutase (odd-number FA)
L-methylmalonyl-coa -> Succinyl-CoA
116
Maple syrup urine disease is caused by?
Deficiency of BCKD (branched-chain alpha-keto dehydrogenase), inability to break down BCAA.
BCKD use the same 5 cofactors as PDH and alpha-ketoglutarate DH complex. Lipoic acid, CoA, NAD, FAD and thyamine pyrophosphate.
117
Steps in BCAA catabolism
1: Branched chain amino acid transaminase (vitB6)
a: Leucine to a-keto-isocapronate
b: Valine to a-keto-isovalerate
c: Isoleucine to a-keto-b-methyl-glutarate
2: Branch-chain alpha-keto acid dehydrogenase complex
a: a-keto-isocapronate to Isovaleryl- CoA -> acetyl-CoA + acetoacetate
b: a-keto-isovalerate to Isobutiryl- CoA -> propionyl-coa
c: a-keto-b-methyl-glutarate to a-methyl-butiryl-CoA -> propionyl-coa + acetyl CoA
118
Glycine cleavage comple: reaction
Glycine + THF + NAD+ ↔ 5,10-methylene THF + CO2 + NH4+ + NADH + H+
119
Choline are formed from?
Serine
120
Taurine are formed from?
Cysteine
121
De novo arginine are made from?
Citruline in the kidney
122
Proline can be formed from
a-KG (glutamate) in a series of reactions.
123
Hyperbilirubinemia lead to?
Jaundice
124
Heme is a prosthetic group in?
Cytochromes, catalase, nitric oxide synthetase, peroxidase, hemoglobin and myoglobin
125
Rate limiting step in porphyrin synthesis
ALA synthase 1 (ALAS1). Inhibitor: Heme
Cofactor: PLP
Glycine + Succinyl CoA -> CoA + Co2 + delta-aminolevulinic acid
126
Chemotherapautic agent which is an inhibitor of dihydrofolate reductase?
Methotrexate
```
DHF reductase is inhibited
No dihydrofolate (DHF) -> Tetrohydrofolate (THF)
```
127
Function of ezetimibe
Blocks the NPC1L1 (Niemann-Pick C1-Like 1)
128
Function of lipoprotein lipase?
Hydrolyzes triglycerides in lipoproteins, such as those found in chylomicrons and very low-density lipoproteins (VLDL), into three free fatty acids and one glycerol molecule.
LPL requires ApoC-II as a cofactor.
LPL is attached to the luminal surface of endothelial cells in capillaries by the protein glycosylphosphatidylinositol HDL-binding protein 1 (GPIHBP1) and by heparan sulfated proteoglycans. It is most widely distributed in adipose, heart, and skeletal muscle tissue, as well as in lactating mammary glands.
LPL isozymes are regulated differently depending on the tissue. For example, insulin is known to activate LPL in adipocytes and its placement in the capillary endothelium. By contrast, insulin has been shown to decrease expression of muscle LPL. Muscle and myocardial LPL is instead activated by glucagon and adrenaline. This helps to explain why during fasting, LPL activity increases in muscle tissue and decreases in adipose tissue, whereas after a meal, the opposite occurs.
129
Hepatic lipase convert .... to ....
IDL to LDL on hepatocytes
130
Dihydrofolate reductase (DHFR) s important in?
The formation of tetrahydrofolate from dihydrofolate. This enzyme use NADPH as a electron donor.
131
Defect in Lesch-Nyhan syndrome
Purine metabolism: deficiency of HGPRT (hypoxanthine-guanine phosphoribosyltransferase)
Guanine + PRPP -> GMP + PPi
Hypoxanthine + PRPP -> Inosine + PPi
Inability to salvage hypo-xanthine and guanine. Excessice uric acid will be formed
132
What is the reaction for the committed step in purine synthesis?
Glutamine:phosphoribosylpyrophosphate amidotransferase
PRPP + Glutamine -> Phosphoribosylamide + PPi + Glutamate
Activator: PRPP
Inhibitor: AMP + GMP
133
Adenine can be converted to AMP by which enzyme?
Adenine:phosphoribosyl transferase
Adenine + PRPP -> AMP + PP
134
Formation of deoxyribonucleotides from ribonucleotides are done by?
Ribonucleotide reductase: Composed of two subunits: R1 + R2 and its specific for the reduction of nucleoside diphosphates (ADP, GDP, UDP, CDP).
The RR is a dimer R1 – catalytic subunit constitutive expression R2 – regulatory subunit induced in the S-phase (E2F transcription factor)
The donor of hydrogens come from two sulfohydryl groups on the enzyme itself. The disulfide bond created after reduction of ribonucleotides are reduced back by Thioredoxin (coenzyme for ribonucleotide reductase). The two sylfohydryl groups on thioredoxin donate their hydrogens to ribonucleotide reductase.
Thioredoxin in converted back to its reduced formed by NADPH + H+ by the enzyme thioredoxin reductase
dATP to its allosteric site inhibit the enzyme
135
Gout is characterized by high levels of?
Uric acid (hyperuricemia)
Hyperuricemia leading to gout can also be seen in fructose intolerance (aldolase B) and von Gierke disease (glucose-6-phosphatase)
136
Purine synthesis (up to inosine monophosphate) need how many ATP, THF derivatives, AA?
```
6 ATP
2 N10-formylTHF
2 Glutamine
Glycine
Aspartate
```
One fumarate is removed in adenosylsuccinate lyase reaction.
137
Which enzymes deficiencies in purine catabolism can cause T-cell deficiency?
Adenosine deaminase (most severe) and purine nucleoside phosphorylase deficiency
Symptoms are: Immunodeficiency.
138
Causes of orotic aciduria
UMP-synthetase (most common) deficiency
| Ornithine transcarbamoylase deficiency
139
How is CTP formed from UTP?
By CTP synthetase
UTP + ATP + Glutamine -> CTP + ADP + Pi + Glutamate
140
Southern, western and nothern blottin check what?
South: DNA
North: RNA
West: Proteins
141
What inhibit glucokinase? What is the disease associated with this transporter?
Fructose 6-P
Glucokinase regulatory protein (GKRP) - fructose 6-P bind to GKRP and remove glucokinase from PM.
Fructose-1P prevent glucokinase removal from PM by binding to GKRP.
Maturity-onset diabetes of the young
Sigmoidal curve
142
Hexokinase is inhibited by?
Glucose 6-P
Hyperbolic curve
143
Glucose 6 phosphatase can be found in which compartment of the cell?
ER lumen
144
Adipose tissue does not express glycerol kinase, how does it make glycerol-3P?
Through glycolysis or glyceroneogenesis (shorter version of gluconeogenesis).
Form DHAP (dihydroacetone-phosphate) -> glycerol 3-P via glycerol-3P dehydrogenase
DHAP + NADH + H+ -> Glycerol-3P + NAD+
This is important for the adipose tissue for the formation of TG.
145
Which is the step in glycolysis where NADH is formed? (reaction)
glyceraldehyde-3P + NAD+ + Pi -> 1,3 bisphosphoglycerate + NADH + H+
Enzyme: Glyceraldehyde 3-P Dehydrogenase
146
What are the names of the substrates and products for the two ATP production steps in glycolysis?
1: Phosphoglycerate kinase
1,3 bisphosphoglycerate + ADP -> 3-Phosphoglycerate + ATP
2: Pyrovate kinase
Phosphoenolpyrovate + ADP -> Pyrovate + ATP
147
What is the steps in the formation and removal of 2,3 bisphosphoglycerate?
1: 1,3 bisphosphoglycerate -> 2,3 bisphosphoglycerate via bisphosphoglycerate mutase enzyme
2: Bisphosphoglycerate phospatase
2,3 bisphosphoglycerate -> 3 phosphoglycerate + Pi
Note: NO substrate level phosphorylation in this pathway
148
What are the parts (catalytic and regulatory) of Pyrovate DH complex? Also include cofactors + reaction
3 metabolic enzymes
E1: Pyrovate dehydrogenase (Thyamine pyrophosphate (TPP))
E2: Dihydrolipoyl transacetylase (lipoic acid)
E3: Dihydrolipoyl dehydrogase (FAD, NAD+ and CoA)
Regulatory units:
1: PDH kinase
2: PDH phosphatase
"Oxidative decarboxylase of pyrovate"
Pyrovate + NAD+ + CoA -> CO2 + acetyl CoA + NADH + H+
High levels of NADH + Acetyl-CoA inhibit E2 and E3
149
What are the activators and inhibitor of PDH kinase and PDH phosphatase
PDH kinase (inhibit PDH activity):
Activators. NADH + Acetyl-CoA, ATP
Inhibitors: NAD+, pyrovate, ADP, AMP, CoA
PDH phosphatase (activate PDH)
Activators: Insulin + Ca2+
Inhibitors: ATP, NADH, Acetyl-CoA
Summary: PDH is stimulated by Ca2+, insulin, NAD+, ADP, and low acetyl-CoA/CoA ratio
Inhibited by NADH, Acetyl-CoA and ATP
150
What are the two reactions in the TCA cycle that remove CO2?
Isocitrate DH and a-ketoglutarate DH
151
Succinate DH is linked to what? what can inhibit this enzyme?
CoQ reductase complex (complex 2). Membrane bound flavoprotein
Malonate competitively inhibit it. Has one carbon (CH2 less than succinate)
152
What are the 3 irreversible reactions in the TCA cycle?
Citrate synthase, Isocitrate DH, a-KG DH complex
153
How many ATP can we get from one acetyl-CoA?
10 acetyl-CoA.
1 GTP (ATP), 3 NADH (3 x 2,5 = 7,5) + 1 FADH (=1,5 ATP)
154
Regulation of citric acid cycle
Citrate synthase:
Activators: ADP
Inhibitors: Citrate, succinyl-CoA, NADH
Isocitrate DH:
Activators: Ca2+, ADP
Inhibitors: ATP, NADH
a-KG DH:
Activators: Ca2+
Inhibitors: Succinyl-CoA, NADH
155
Aldolase B is associated with which disease and what does it catalyse?
Fructose intolerance
Fructose 1-P -> DHAP + Glyceraldehyde
156
What is the problem with Wernicke-Korsakoff syndrome?
Lack of transketolase or thyamine pyrophosphate
157
What are the 3 diseases we can find in galactose metabolism?
Type 1: Galactose 1-P uridyltransferase
Type 2: Galactokinase
Type 3: UDP-galactose 4-epimerase
158
What is the disease associated with fructokinase deficiency?
Essential Fructosuria
159
What is wrong in the 5 first glycogen diseases and what is their name?
Type 1: von gierke: Glucose 6-phosphatase (a) or glucose 6-P translocase (b)
Type 2: Pomp disease: Lysosomal glucosidase
Type 3: Cori disease: Debranching enzyme
Type 4: McArdle disease: Muscle phosphorylase
Type 5: Andersons disease: Branching enzyme (in liver)
160
Hemolytic anemia can be caused by?
Pyrovate kinase or glucose-6P DH (this is called drug induced hemolytic anemia) deficiency
161
Reaction and disease associated with prolyl hydroxylase (procollagen-proline dioxygenase)?
Scurvy
Proline + O2 + a-KG -> 4-Hydroxy-proline + CO2 + Succinate
Fe2+ Vitamin C
Lysyl hydroxylase do the same reaction with lysine
162
What is the common primary structure in collagen?
Gly-X-Y where X and Y is proline and hydroxyproline
Hydroxylysine can also be in this sequence
163
What is the sequence of steps from procollagen to mature collagen?
Procollagen is cleaved by procollagen peptidases to remove the glubular heads of the N- and C-terminal. This forms tropocollagen. Tropocollagen polymerization -> Lysyl oxidase -> form bridges between tropocollagen fibers (Lysinonorleucin bridge)
Lysyl oxidase require Cu2+ and molecular O2
Lysine + O2 -> NH3 + H20
164
What are the two diseases associated with collagen?
Ehlers-Danlos syndrome: Deficiency of lysyl hydroxylase or procollagen peptidase. Very stretchable skin.
Osteogenesis imperfecta: Procollagen (triple helix) formation is incomplete. Fraglie bones.
165
What is a protein Domain?
a section of the protein structure for a particular task
| Multidomain proteins
166
Name one specific inhibitor of serine enzymes
Diisopropyl fluorophosphate (DIFP)
167
Name 3 lipases in adipose tissue (which one is regulated)?
HSL (regulated by phosphoryation): remove the first FA
Diacylglycerol lipase
Monoacylglycerol lipase
Perilipin blocks these enzymes. Phosphorylation remove this inhibition.
168
How is FFA "activated" for oxidation? Which cellular compartment does this occur?
In cytosol
Fatty Acyl-CoA Synthetase
ATP + FA + CoA -> AMP + PPi + Fatty Acyl-CoA
(Acyl-Adenylate intermediate)
We use 2ATP!
169
Carnitine acyl transferase 1 (CAT/CPT1) is the rate-limiting step in what and inhibited by?
Rate-limiting step in the transport of Fatty-acyl-CoA to the mitochondria for beta-oxidation.
Malonyl-CoA inhibit
Acyl-CoA + carnitine -> Acyl-carnitine + CoA
170
Which 2 enzymes do we need to make TG from 2-monoacylglycerol?
Acyl-CoA:monoacylglycerol acyltransferase
| Acyl-CoA:Diacylglycerol acyltransferase
171
What are the 3 basic AA and what is special with one of them?
Histidine, arginine and lysine
Arg + lys have a positive charge on its R-group on its isoelectric point.
Histidine has NO charge on its R-group at iP, but it does have a positive charge on its alpha amino group at iP. alpha-amino has lower pK that its R-group.
172
Which AA has two and zero chiral groups?
Two: Threonine and isoleucine
Zero: Glycine
173
What is the most common genetic defect in fatty acid oxidation? What is the treatment?
Deficiency of medium chain acyl-CoA dehydrogenase
Carbohydrate rich meals and carnitine supplementation
174
Carnitine can be made from two AA, which?
Lysine and methionine
Occur in the liver and kidney
175
What are the recommended tretment in carnitine deficiencies?
Avoid LCFA and long fasts
Take: carnitine supplements, MCFA, diet rich in carbohydrates
176
What are the steps in B-oxidation?
1: Acyl-CoA dehydrogenase (FADH gainz)
2: Enoyl-CoA hydratase (Feed me water!)
3: Beta-Hydoxyacyl CoA Dehydrogenase (NADH gainz)
4: Beta-ketoacyl-CoA thiolase (or Acyl-Coa-acetyltransferase) (Come to daddy CoA)
177
How many acetyl-coa, NADH and FADH do we get from one palmitoyl-coa?
8 acetyl-coa, 7 NADH and 7 FADH2
129 ATP in total after activation
Activation of palmitate require 2 ATP
178
Oxidation of odd-number fatty acids need which enzymes (also give the names of the intermediates)?
Propionyl-CoA -> D-methylmalonyl-coa -> L-methylmalonyl-coa -> Succinyl-coa
Enzymes:
1: Propionyl-CoA carboxylase (biotin, co2 and ATP)
2: Methylmalonyl coa racemase
3: Methylmalonyl coa mutase (vitb12)
179
What are the names of the intermediates in ketone body synthesis? (+enzymes)
Acetyl-Coa -> Acetoacetyl-CoA -> HMG-CoA -> Acetoacetate -> B-hydroxybutyrate or Acetone
Enzymes:
Thiolase -> HMG coa synthase -> HMG CoA lyase -> B-hydroxybutyrate DH.
HMG-CoA synthase is the rate-limiting step
180
Creatine is made from which AA and in which organs?
Glycine and arginine. Liver (first) and kidney (secondary, get SAM in its ass)
Use SAM as a methyl donor
181
Which steps in ketone body synthesis and degradation are different?
Acetoacetate to Acetoacyl-CoA step.
Use Succinyl-CoA as a CoA donor.
Acetoacetate + Succinyl-CoA -> Acetoacyl-Coa + Succinate
Via beta-ketoacyl CoA transferase/thiophorase
182
Fatty acid synthase has which fucker attached to is?
4-Phophopantetheine
183
Reaction equation for malic enzyme
Malate + NADP+ -> Pyrovate + NADPH + H+ + CO2
184
What is the rate-limiting and regulated step in cholesterol synthesis? What is the reaction?
HMG CoA reductase (found in ER)
HMG CoA + 2 NADPH + 2 H+ -> Mevalonate + CoA + 2 NADP+
185
How many ATP, NADPH, Acetyl-CoA and glucose do we use to make 1 cholesterol molecule?
ATP: 36
Acetyl-CoA: 18
NADPH: 20
Glucose: 9
186
Which part of cholesterol synthesis makes a head-head reaction?
Farnesyl PP -> Squalene
187
What is the function of SCAP?
cholesterol binds to SCAP (SREBP cleavage-activating protein). cholesterol sensor-SREBP is bound to SCAP -> SREBP is inactive
Sterol regulatory element-binding proteins (SREBPs,) are embedded in the ER when first synthesized, in a complex with the protein SREBP cleavage-activating protein (SCAP). (N and C represent the amino and carboxyl termini of the proteins.) When bound to SCAP, SREBPs are inactive. When sterol levels decline, the complex migrates to the Golgi complex, and SREBP is cleaved by two different proteases in succession. The liberated amino-terminal domain of SREBP migrates to the nucleus, where it activates transcription of sterol-regulated genes.
188
What is the name of the protein that regulate LDL-R in the liver?
PCSK9. This protein bind to LDL-R and promote degration.
189
What binds and activate LXR?
Oxysterol
190
What is IDOL and what regulate it?
Activated by LXR. Increased Degradation of LDL Receptor Protein (IDOL) is a ubiquitin ligase that ubiquinates LDL receptors in endosomes and directs the receptors to the lysosomal compartment for degradation. IDOL is transcriptionally up-regulated by LXR/RXR in response to an increase in intracellular cholesterol.
The expression of IDOL is induced by the sterol-ativated liver X receptor.
Remove LDL-R when cholesterol is high intracellulary
191
Insig: function?
INSIG1 plays an important role in the SREBP-mediated regulation of cholesterol biosynthesis: by binding to the sterol-sensing domain of SCAP (SREBP cleavage activating protein) it makes the SCAP/SREBP complex stay longer in the ER, thus prohibiting SCAP from carrying activated SREBP to the golgi complex. This ultimately blocks SREBP from acting as a transcription factor for the SRE in the promoter region of the HMG-CoA-reductase gene and results in a decreased expression of HMG-CoA-reductase.
INSIG1 also binds to the sterol-sensing domain of HMG-Co-A-reductase, resulting in the enzyme's increased degradation.
192
Give me some names of primary bile acids. What can they be conjugated with? Where is the hydroxy groups?
Cholic acid (3,7,12 and chendodeoxycholic acid (3,7)
Taurine and glycine
Glycocholic acid
Taurocholic acid
Taurochendodeoxycholic acid
Glycochendodeoxycholic acid
193
Name some secondary bile acids and hydroxyl groups are placed?
```
Deoxycholic acid (3,12)
Lithcholic acid (3)
```
194
Main ligand for FXR
Bile acids
195
CAD complex consists of which 3 enzymes and are regulated by what?
Carbamoyl phosphate synthetase 2
Aspartate carbamoyltransferease
Dihydroorotase
+ regulation: PRPP
- regulation: UTP (negative feedback
196
UMP synthase complex consists of which two enzymes? What disease is caused by a defect in this complex?
Orotate phosphoribosyl transferase
OMP decarboxylase
- Regulation by UMP
Orotic aciduria
197
What is the apropriate rate of ion transport through a ion channel?
The rate of ion transport through the channel is very high, often 10^6-8 ionsper second. This rate is approaching the theoretical maximum for unrestricted diffusion! Ion channels is not saturable (under normal physiological conditions). It is theoretically possible to saturate ion channels. Experiments with supraphysiological concentration of ions saturate ion channels. o They are gated in response to some cellular event. E.g. ligand, voltage, mechanical
198
Open probability (Po) of a channel can be defined as
Po = Total time in the open state / Total time observed
199
What determine the selectivity of K+ and voltage gated Ca2+ and Na+ channels?
P-loop
200
The voltage sensor in the K+-channel is?
S3-S4-helix-turn-helix
The structure of the mammalian voltage-gated K+ channel has been used to explain its ability to respond to the voltage across the membrane. Upon opening of the channel, conformational changes in the voltage-sensor domains (VSD) result in the transfer of 12-16 elementary charges across the membrane electric field. Several charged residues of the VSD, in particular four arginine residues located regularly at every third position on the S4 segment, are known to move across the transmembrane field and contribute to the gating charge
201
Explain "N-type" or "Ball in chain" inactivation
the N-terminus of the protein forms a ball that is tethered to the rest of the protein through a loop (the chain). The tethered ball blocks the inner porehole, preventing ion movement through the channel
In cellular neuroscience ball and chain inactivation is a model to explain the fast inactivation mechanism of voltage-gated ion channels. The process is also called hinged-lid inactivation or N-type inactivation. A voltage gated ion channel can be in three states: open, closed or inactivated. The inactive state is mainly achieved through fast inactivation, by which a channel transitions rapidly from an open to an inactivated state. The model proposes that the inactive state, which is stable and non-conducting, is caused by the physical blockage of the pore. The blockage is caused by a ball of amino acids attached to the main protein by a string of residues on the cytoplasmic side. The ball enters the open channel and binds to the hydrophobic inner vestibule at the center of the channel. The blockage causes inactivation of the channel by stopping the flow of ions
202
What are ATP-sensitive K+ channels and can you give any general information about it?
An ATP-sensitive potassium channel (or KATP channel) is a type of potassium channel that is gated by intracellular nucleotides, ATP and ADP. ATP-sensitive potassium channels are composed of Kir6.2-type subunits and sulfonylurea receptor (SUR) subunits, along with additional components.
Octamer: 4 SUR + 4 Kir.6.2. SUR have 3 TM domains and 2 nucleotide binding domains (NTBD). These allow for nucleotide-mediated regulation of the potassium channel, and are critical in its roles as a sensor of metabolic status.
ATP blocks these channels by binding to NTBD and cause depolarization. Important in the regulation of insulin
Normally, the concentration of ATP in the cell is so high that is would inhibit practically all KATP channels. The normal ATP concentration in a cell is in the mM range. The ATP concentration required to inhibit half of its activity is 14 μm. The only way for this channels to be open is to have activators such as ADP. So, in short, this channels are sensitive to the ATP/ADP ratio.
ATP act on Kir6.2
ADP, sulfonylureas work on SUR
Pharmacology:
Inhibitor: Sulfonylurea
Activators: Diazoxide
203
What is special with voltage-gated Cl- channels?
Each channel or exchanger is composed of two similar subunits—a dimer—each subunit containing one pore. Each subunit binds ions independently of the other, meaning conduction or exchange occur independently in each subunit. Each subunit consists of two related halves oriented in opposite directions, forming an ‘antiparallel’ structure. These halves come together to form the anion pore. The pore has a filter through which chloride and other anions can pass, but lets little else through. Due to the positive charge in its selective part, cations such as K+, Na+ and Ca2+ are repelled.
204
Explain what CFTR is? regulation ++
CFTR functions as an ATP-gated anion channel (not a pump!), increasing the conductance for certain anions (e.g. Cl−) to flow down their electrochemical gradient. CFTR is an ion channel that evolved as a 'broken' ABC transporter that leaks when in open conformation. We use energy to keep the channel open.
CFTR consists of a single polipeptide chain with two 6 transmembrane helixes (MSD1 and MSD2): 2 transmembrane domains (TMD), 2 intracellular nucleotide binding domains (NBDs), and a regulatory (R-) domain.
The channel conducts Cl- across the plasma membrane, only when both ATP are bound to NBDs. This opening continues until ATP of one of the NBDs is broken down to ADP + Pi.
The CFTR Cl- channel is phosphorylated on at least 10 Ser residues by PKA)on its regulatory domain; phosphorylation is a prerequisite for activation. The channel does NOT open without this phosphorylation. Channel activity incrementally increases with phosphorylation.
Gating of phosphorylated channels requires the presence of intracellular ATP.
The active conformation of ABC proteins are head-tail NBD1 and NBD2. NBD2 hydrolyse ATP which causes the dimer to dissociate and the pore close.
205
Nicotinic Ach receptor: structure
Nonselective cation channel. It consists of a pentameric channel comprising four different homologues subunits. The nAchR are activated when two ligands bind to the clefts between the α and δ in one part and between α and γ (ε in adult).
In the muscle-type receptors, found at the neuromuscular junction, receptors are either the
Embryonic form: composed of 2α, β1, γ, and δ subunits in Adult form composed of 2α, β, δ, and ε subunits
Neural type: 2α, 3β or only 5α
206
What is the name of the disease which nicotinic Ach receptors in the neuromuscular junction is not function properly?
Myasthenia gravis
207
Name some P-type ATPases
1: Na/K ATPase
2: SERCA
3: K/H ATPase (exchanger)
4: Plasma membran Ca2+ ATPase (PMCA)
5: H+-ATPase (proton pump)
208
What is the function of the FXYD unit (phospholemman in the heart) on the Na/K pump?
The FXYD unit is the “third subunit” in the Na-K-pump. Its only present on some tissues such as the heart and kidney. Regulate the catalytic function of the alpha subunit. The function of the Na-K-ATP is done by the alpha unit. Beta unit stabilize the alpha unit, due to the highly glycosylated region on its extracellular surface. Prevent flipping of the protein complex.
The presence of FXYD1 in the heart decreases its affinity to Na+ à higher Na+ concentrations are required for the Na-K-pump to switch on à Higher Na+ intracellulary à NCX activity increase and exchange Na+ with Ca2+ à More Ca2+ in the cell.
After beta 1 stimulation à PKA à FXYD1 unit phosphorylated à increase Na+ affinity on alpha unit à lower intracellular Na+ levels and reduced NCX activity. Prevent arrhythmia.
In the kidney. The presence of FXYD2 increase the affinity to ATP. Allow Na-K-pump to function even when the ATP concentration is low. This is particularly important in kidney medulla which work under anoxic conditions. Fine tune the activity of Na-Kpump.
209
What is the name of the inhibitor of Na/K pump? What are its effect on the heart?
Ouabain. Bind to E2P
Ouabain is a cardiac glycoside that acts by inhibiting the Na+/K+-ATPase sodium-potassium ion pump. Once ouabain binds to this enzyme, the enzyme ceases to function, leading to an increase of intracellular sodium. This increase in intracellular sodium and increase the activity of the sodium-calcium exchanger (NCX), which pumps one calcium ion out of the cell and three sodium ions into the cell down their concentration gradient, just that now it work in reverse, transporting Na+ out of the cell and Ca2+ into the cell. Therefore the decrease in the concentration gradient of sodium into the cell which occurs when the Na/K-ATPase is inhibited reduces the ability of the NCX to function. This in turn elevates intracellular calcium. This results in higher cardiac contractility (positive inotropic effect)
210
What are the Na/K pump sensitive to (regulation)?
Na+ concentration and ATP (no role in normal physiological concentration. Always saturated)
211
Function of vesiclular monoamine transporter
| VMAT
```
Vesiclular monoamine transporter
(VMAT) is used to transport
neurotransmitter molecules from the
cytoplasm of the cell to the interior of
the synaptic vesicles
```
212
SERCA is regulated by what molecule (in the heart)?
Phospholamban in the heart. SERCA is normally inhibited by PLB, with which it is closely associated. Increased β-adrenergic stimulation reduces the association between SERCA and PLB by the phosphorylation of PLB by PKA. When PLB is associated with SERCA, the rate of Ca2+ movement is reduced; upon dissociation of PLB, Ca2+ movement increases.
213
What is the reaction and name of the enzyme that forms acetylcholine
Choline acetyltransferase
Acetyl-CoA + choline -> CoA + Ach
214
What is the name of the high affinity choline uptake transporter?
Na+dependent choline uptake in neurons.
Inhibitor: Hemicholinium
215
What is the name of the molecule that function as a Ca2+-sensor in exocytosis?
Synaptotagmin (on its C2 domain)
216
What is the name of the drug that inhibit vesicular exocytosis of Ach?
Botulinium toxin.
Inhibit the exocytosis in the light chain domain (catalytic). Cleaves the Zn2+-dependent endoproteases, absolute specific for certain sites for the SNARE proteins.
217
E1 and E2 in Na/K ATPase has which affinity differences?
E1: Higher affinity for Na and ATP
E2: Low affinity for ATP
218
Name of the general inhibitor of nAch receptor?
Curare
219
Name two drugs that are specific for muscle nAch receptors
D-tubocuraine and a-bungarotoxin
220
Name one specific inhibitor of muscarinic Ach receptor
Atropine
221
Muscarinic receptors: M1, M3 and M5 are coupled with?
Gq, (PLC activation, activation of adenylate
| cyclase (cAMP), and activation of K+-channels.)
222
Muscarinic receptors: M2 and M4 are coupled with?
Gi
223
Ryanodine isoform 1 and 2 can be found where?
RYR1 in skeletal muscle
RYR2 in hear muscle. Imporant in calcium-induced calcium release. Phosphorylation of these channels increases their permeability to calcium and increases the contractility of their respective cardiac myocytes.
224
Malignant hyperthermia is most ofted caused by a mutation in?
RYR1 in muscles
Can also be caused be exposure to certain drugs used for general anesthesia — specifically the volatile anesthetic agents and succinylcholine, a neuromuscular blocking agent
225
Adrenergic receptor are coupled with what?
alpha 1: Gq
alpha 2: Gi
beta 1,2,3: Gs
226
Epinephrine work on which receptors in adipose tissue? what is its effects?
Beta 1 and 3 both are Gs coupled -> PKA -> Phosphorylation of perilipin and HSL.
PKA stimulate mobilization of glycogen and inhibition of glycogen synthesis by phosphorylation of glycogen phosphorylase kinase and glycogen synthase, respectively.
Adipose tissue also has α1 and α2 receptors. α1 stimulation is Gq coupled à CaM kinase à mobilization of glycogen and inhibition of glycogen synthesis. Almost the same as with beta stimulation, but without lipolysis. α2 receptors on the other hand has the opposite action on adipose tissue. These receptors are Gi coupled which lowers cAMP and inhibit lipolysis. Since the amount of beta receptors is much higher than alpha receptors, the overall effect will be lipolysis in adipose tissue. Might be some differences in the relative amount of each receptor in different locations in the body. The amount of α2 receptors can be increased during fasting.
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What is epinephrine`s effect on skeletal muscle?
Epinephrine stimulate β2 receptors in skeletal muscle à cAMP increase à PKA. Glycogen synthesis stops while glycogen breakdown increase. Glycogen synthase is phosphorylated by PKA à inactivated à reduced glycogen synthesis
Extra: Ca2+ act as an allosteric activator, binding to the δ subunits of phosphorylase kinase, and partly activate the enzyme. This binding partly stabilizes the protein in its active form. Glycogen phosphorylase kinase is completely activated when the β and α subunits are phosphorylated by PKA and the delta subunit has bound to 4 calcium ions. .
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What is the molecules, cofactors and enzymes in the formation of epinephrine from tyrosine? Regulations
Enzymes
1: Tyrosine hydroxylase (rate-limiting)
2: L-DOPA decarboxylase (aka aromatic amino acid decarboxylase)
3: Dopamine-beta-hydroxylase
4: Phenyl-ethanolamine-N-methyltransferase
Tyrosine + O2 + Tetrahydroptherin -> L-DOPA + H2O + Dihydroptherin
L-DOPA -> Co2 + Dopamine (PLP dependent)
Dopamine + O2 -> Norepinephrine + H20 (Vitamin C + Cu2+)
Norepinephrine + SAM -> Epinephrine + SAH
Tyrosine hydroxylase is regulated be Ca2+, PKA, PKC CaM kinases. Negative feedback by its norepinephrine.
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Where in the cellular compartment are dopamine, norepinephrine and epinephrine made?
Dopamine is synthesized in cytosol. Dopamine is taken up by the vesicles. If there is no dopamine-beta-hydroxylase here, then it will be stored in this vesicles. These are the dopaminergic neurons. In adrenal gland and in noradrenergic neurons the vesicles do have this enzyme and convert dopamine to NE. NE “leaks” out of the vesicular membrane. The leaked NE are taken up by transporters into the vesicles again. This leakiness is important because the enzyme which converts NE to E is located in cytosol. Epinephrine synthesized in cytosol is taken up by the vesicles.
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Which partydrug compete with catecholamine uptake?
Amphetamine compete with endogenous catecholamine uptake. Inhibits both vesicular monoamine transporters, VMAT1 and VMAT2
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Function of Vesicular monoamine transporter (VMAT)
integral membrane protein, which is embedded in synaptic vesicles and serves to transfer monoamines, such as norepinephrine, epinephrine, dopamine, and serotonin, between the cytosol and synaptic vesicles
VMAT1 and VMAT2
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Function of monoamine oxidases (MAO) is catecholamine metabolism
Monoamine oxidases (MAO) catalyse the oxidative deamination of monoamines. Oxygen is used to remove an amine group from a molecule, resulting in the corresponding aldehyde and ammonia. Monoamine oxidases contain the covalently bound cofactor FAD and are, thus, classified as flavoproteins. MAO is present in the outer mitochondrial membrane. We have two main groups om MAO:
MAO-A: In neurons and in the periphery (preferance for norepinephrine and 5-HT)
MAO-B: In neurons (substrate preferance for dopamine. Important in parkinsons disease)
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General reaction of MAO
X + FAD -> Y + NH3 + FADH2
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What would you give to a patient with parkinsons disease?
MAO B inhibitor: Deprenyl
| L-DOPA to increase dopamine synthesis.
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What is COMPT (catechol-O-methyltransferase) and its function?
Catechol-O-methyltransferase is involved in the Inactivation of the catecholamine neurotransmitters (dopamine, epinephrine, and norepinephrine). The enzyme introduces a methyl group to the catecholamine, which is donated by S-adenosyl methionine (SAM). Any compound having a catechol structure, like catechol-estrogens and catechol-containing flavonoids, are substrates of COMT. This enzyme is present intra and extracellularly.
236
What are the endproducts of noradrenaline catabolism in the CNS and periphery?
3-methoxy-4-hydroxyphenyl glycerol (MHPG) is the main end-product in the CNS
3-methoxy-4-hydroxymandelic acid (VMA) is the main end-product in the periphery.
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Norepinephrine transport system (NET)
the neurotransmitter it taken up (reuptake) in the Presynaptic neuron. This is how the neurotransmitters are cleared from the synaptic cleft. This uptake is done by NET (norepinephrine transport system), an Na+-Catecholamine cotransporter which uses the electrochemical gradient of Na+ to transport catecholamines into the cell (secondary active transport). The transporter can work in reverse if the concentration gradient is favourable to move Na+ and catecholamine into the extracellular fluid.
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Name two inhibitors of NET (norepinephrine transport system)
Cocaine and amphetamine (competitive)
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Inhibitor of dopamine-decarboxylase
alpha-methyl-DOPA
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Tyrosine hydroxylase inhibitor
alpha-methyl-tyrosine
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What is the end product of dopamine metabolism?
What are the 3 enzymes
Homovalilic acid
decreased in parkinsons and increased in schizophrenia
1: MAO-B
2: Aldehyde dehydrogenase
3: COMT (SAM)
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What is the function of guanylate cyclase, what is its reaction and what kind of types do we have? What can activate guanylate cyclase?
Soluable and membrane bound (activated by ANF)
GTP -> cGMP + PPi
ANF -> Aldosterone and ADH/vasopressin secretion inhibition. Vasodilation. Na+ excretion.
Activators: NO activate soluable GC. ANF activate membrane bound GC.
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Reaction of Glutamic acid decarboxylase/Glutamate decarboxylase. What is its cofactor?
PLP
Glutamate -> Co2 + GABA
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Reaction of GABA transaminase
GABA + a-KG -> Glutamate + succinic semialdehyde (SSA)
Succinic semialdehyde are broken down to succinate via succinic semialdehyde dehydrogenase (we gain NADH)
245
Which one activate (remove Mg2+ inhibitor) from the other receptor. NMDA or AMPA
AMPA first -> Mg2+ block is removed by depolarization -> NMDA opens
246
GABA1 and GABA2 receptors are?
GABA1: Inotropic
GABA2: Metabotropic
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SH2 and SH3 binds?
SH2: phosphorylated tyrosine residues
SH3: binds to proline rich regions (E.g. SOS)
SH = Src homology domains
Work as docking proteins
248
Insulin recetor consists of which subunits?
2 extracytoplasmic alpha unit with one binding site for insulin each. Upon binding, alpha unit changes its conformation an activate its two beta subunits.
Hetrotetramer (2alpha, 2 beta)
Conformation change activates b-subunit TK activity, without ligand allosteric inhibition of TK activity. b subunit phosphorylates Tyr residues on cytoplasmic domains as well as downstream substrates (IRS). IRS:insulin receptor substrate is also phosphorylated by IR
249
Insulin can activate which pathways?
PI3-K: Phosphatidylinositol 3-kinase, makes PIP2,PIP3 – protein kinase B /Akt/ activation metabolic changes
Grb2, Sos, activates Ras – MAP kinase pathway
Activation of PI-PLC
250
Most RTK are present as a ... in its inactive form. What hapens when it ligand binds?
Monomer in its inactive state. Dimerize after ligand binding.
251
What are the proteins involved in MAPK pathway?
Ras -> Raf -> MEK1 -> ERK 1
ERK1 phosphorylate transcription factors
252
Cytokines work on which cellular pathway?
JAK-STAT. Cytokine receptor is a hetrodimer.
253
Steriod hormones form a ... on its HRE (Hormone response element)?
Homodimer
254
Non-steriod hormones forms a ... on its HRE (hormone response element)?
Hetrodimer
No HSP associated with them. Can bind to HRE as a monomer without ligands. Has a silencer effet.
255
Glucocorticoid receptor is normaly bound to what in its inactive form?
HSP90. Dissociate after ligand binding.
Similar with other steroid hormone receptors
No HRE binding without ligand but repressor effects. Must dimerise for HRE binding. No silencer effect.
256
AHR (acryl hydrocarbon) are induced by?
Polyaromoatic hydrocarbons
257
HIF1-a and AHR are clasified as? (family)
bHLH (basic helix-loop-helix). Hetrodimers
258
HIF1-a is bound to what?
HIF1-b -> Forms a dimer. HIF-1a is oxygen sensitive, HIFF1-b is not O2 sensitive.
259
How is HIF-1a degraded
ODD (oxygen dependent degradation). O2 present -> 4-hydroxyproline formation (by proyl-hydroxylase (Fe2+ and O2)) -> targeted by ubiquitylation by Von-Hippel Lindau protein (E3-ligase).
In hypoxia, HIF-1 are not hydroxylated on proline residues -> act on hypoxia response element (HRE) on DNA, still as a hetrodimer.
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What is the function of Von-Hippel Lindau protein?
The VHL protein (pVHL) is involved in the regulation of a protein known as hypoxia inducible factor 1α (HIF1α). This is a subunit of a heterodimeric transcription factor that at normal cellular oxygen levels is highly regulated. In normal physiological conditions, pVHL recognizes and binds to HIF1α only when oxygen is present due to the post translational hydroxylation of 2 proline residues within the HIF1α protein. pVHL is an E3 ligase that ubiquitinates HIF1α and causes its degradation by the proteasome. In low oxygen conditions or in cases of VHL disease where the VHL gene is mutated, pVHL does not bind to HIF1α. This allows the subunit to dimerise with HIF1β and activate the transcription of a number of genes, including vascular endothelial growth factor, platelet-derived growth factor B, erythropoietin and genes involved in glucose uptake and metabolism
261
Why is RXR so important?
RXR heterodimerizes with subfamily 1 nuclear receptors including CAR, FXR, LXR, PPAR, PXR, RAR, TR, and VDR.
Hetrodimers are needed for its activity
262
Asparagine hydroxylation by TIH on HIF1-a is responsible for what?
inactivation of P300 (HAT activity). Block coactivator recruitment.
P300/CBP = coactivator protein of HAT activity
263
Signal transduction: What is the most most conserved domain?
DNA-binding domain (2 Zinc fingers -> Form a dimer)
264
What initiate repilcation in prokaryotes?
DnaA, it binds to oriC region
265
What is the function of Dam methylase?
DAM methylase, an abbreviation for Deoxyadenosine methylase, is an enzyme that adds a methyl group to the adenine of the sequence 5'-GATC-3' in newly synthesized DNA. Immediately after DNA synthesis, the daughter strand remains unmethylated for a short time.
A repair enzyme, MutS, binds to mismatches in DNA and recruits MutL, which subsequently activates the endonuclease MutH. MutH binds hemimethylated GATC sites and when activated will selectively cleave the unmethylated daughter strand, allowing helicase and exonucleases to excise the nascent strand in the region surrounding the mismatch. The strand is then re-synthesized by DNA polymerase III.
