BIOCHEMISTRY FINAL REVIEW Flashcards
(312 cards)
1
Q
Only amino acid with an R absolute configuration
A
Cysteine, still qualifies as an L-amino acid though
2
Q
pKa for carboxyl group of amino acid
A
usually around 2
3
Q
pKa for amino group of amino acid
A
between 9 and 10
4
Q
amino acid charge under acidic conditions and basic conditions
A
AAs are positively charged under acidic conditions, negatively charged under basic conditions -acidic AAs are negatively charged, basic AAs are positively charged
(under physiological pH the acidic group is removed on acidic amino acids)
5
Q
Calculating pI
A
pI of an acidic amino acid = average two most acidic groups pI of a basic amino acid = average two most basic groups
6
Q
Peptide bond formation, rotation, and hydrolysis
A
peptide bond formation is a condensation or dehydration reaction between an amino terminus and a carboxy terminus
no rotation because of partial double bond character
in living organisms hydrolysis is catalyzed by trypsin and chymotrypsin
in organic chemistry hydrolysis can be catalyzed by acids or bases
7
Q
Proline effects on DNA
A
Because of its rigid cyclic structure, proline will introduce a kink in the peptide chain when it is found in the middle of an alpha helix however, in beta-pleated sheets it is found in the turns
8
Q
Tertiary structure interactions
A
Tertiary structure is determined by hydrophilic and hydrophobic interactions between R groups, as well as hydrogen bonding and acid-base interactions between amino acids with charged R groups, and disulfide bonds
9
Q
Disulfide bonds
A
Disulfide bonds form from two cysteines oxidized to cystine create loops in the protein chain, determine how wavy hair is forming requires the loss of two protons and two electrons (oxidation)
10
Q
Quarternary structures special characteristics
A
Quaternary structures can induce cooperativity or allosteric effects
11
Q
Conjugated proteins
A
Conjugated proteins derived part of their function from covalently attached prosthetic groups Lipoproteins, glycoproteins, and nucleoproteins are named differently due to the type of prosthetic group
12
Q
Oxidoreductases
A
oxidation-reduction reactions, the transfer of electrons Includes dehydrogenases, reducatases, and oxidases
13
Q
Transferases
A
catalyze the movement of a functional group includes kinases, which transfer a phosphate group
14
Q
Hydrolases
A
catalyze hydrolysis (cleavage of a compound using water)
15
Q
Lyases
A
catalyze the cleavage of a single molecule into two products without water
16
Q
Isomerases
A
Rearrange the bonds within a molecule Some can also be classified as oxidoreductases, transferases, or lyases
17
Q
Ligases
A
catalyze addition or synthesis reactions encountered in nucleic acid synthesis and repair
18
Q
Cofactors and Coenzymes
A
Cofactors are generally inorganic molecules or metal ions, coenzymes are small organic groups derived from vitamins
Participate in the catalysis of the reaction, usually by carrying charge through ionization, protonation, or deprotonation
Enzymes without their cofactors are called apoenzymes, whereas those containing them are holoenzymes
Tightly bounds ones are known as prosthetic groups
19
Q
Michaelis-Menton plot and equation
A
A Michaelis-Menton plot relates velocity to concentration
v = (vmax*[S]) / (Km + [S]) velocity of reaction =
(maximum velocity * concentration) / (concentration at half vmax + concentration)
Km is known as the Michaelis constant, lower Km = higher affinity
20
Q
Lineweaver-Burk
A
A Lineweaver-Burk plot is a double reciprocal of the Michaelis-Menton equation
The y-intercept is the reciprocal of Vmax, the x-intercept is the reciprocal of Km
Both Vmax and Km increase in value as they go towards the origin
21
Q
Cooperativity enzyme kinetics; Hill’s coefficient
A
show a sigmoidal relationship on a Michaelis-Menton plot
Binding of a substrate encourages the transition of other subunits from the T(tense) state to the R(relaxed) state
Hill’s coefficient quantifies cooperativity; >1 = positively cooperative, <1 = negatively cooperative
22
Q
Effect of Temperature on enzyme kinetics
A
enzyme-catalyzed reactions tend to double in velocity for every 10C until optimal temperature is reached, for the human body, this is 37C
23
Q
Effect of pH on enzyme kinetics
A
optimal pH is 7.4 for most enzyme catalyzed reactions
24
Q
effect of Salinity on enzyme kinetics
A
Increasing levels of salt can disrupt hydrogen and ionic bonds
25
Feedback regulation enzymes
enzymes being subject to the products of their reactions
negative feedback
opposite is forward regulation
26
Competitive inhibition
Occupies the active site
Does not change Vmax but increases Km
27
Noncompetitive inhibition
Occupies the allosteric site
Decreases Vmax but does not change Km
28
Mixed inhibition
Inhibitor can bind to either enzyme or the ES complex, but has different affinity for each
Bind to allosteric site
Alters the Km depending on the preference of the inhibitor for the enzyme vs the enzyme-substrate complex
- if it binds to the enzyme, it increases the Km
- if it binds to the complex, it lowers Km
- always decreases the Vmax
29
Uncompetitive inhibition
binds to the ES complex and locks the substrate in the enzyme
-can be interpreted as increasing affinity
Lowers Km and Vmax
30
Irreversible inhibition
the active site is made unavailable or the enzyme is permanently altered
-a prime drug mechanism
31
allosteric enzymes
Alternative between an active or inactive form
Molecules that bind may either be activators or inhibitors
32
covalently modified enzymes
Enzymes can be activated or deactivated by phosphorylation or dephosphorylation
33
collagen
has a characteristic trihelical fiber and makes up most of the extracellular matrix of connective tissue
34
elastin
another important component of the extracellular matrix primary role is to stretch and recoil
35
keratin
intermediate filament proteins found in epithelial cells contribute to the mechanical integrity of the cell makes up hair and nails
36
actin
makes up microfilaments and the thin filament in myofibrils most abundant protein in eukaryotes have a positive side and a negative side allows motor proteins to travel unidirectionally along
37
tubulin
makes up microtubules
important for structure, chromosome separation, intracellular transport using kinesins and dyneins
38
myosin
primary motor protein that interacts with actin
39
kinesins and dyneins
motor proteins associated with microtubules have two heads, one of which remains attached to tubulin at all times
align chromosomes, bring vesicles toward one end
positive-kinesins, negative-dyneins
40
examples of binding proteins
hemoglobin, calcium-binding proteins, DNA-binding proteins, etc
41
Cell adhesion molecules
CAMs proteins found on the surface of most cells and aid in binding the cell to the extracellular matrix of other cells (Ex: cadherins, interns, selections)
42
cadherins
group of glycoproteins that mediate _calcium-dependent_ cell adhesion
usually hold similar cells together, such as epithelial cells
_bind to each other_
43
integrins
have two membrane-spanning chains called alpha and beta
binding and communicating with the _extracellular matrix_
cellular signalling can promote cell division, apoptosis, or other processes
44
selectins
bind to carbohydrate molecules that project from other cell surfaces
weakest bonds formed by the CAMs
expressed on white blood cells and the endothelial cells that line blood vessels
45
Antibody three functions
Neutralize - unable to exert its effect
Opsonize - mark for destruct
Agglutinate - clump together
46
Ion channels - ungated channels
no gates and therefore unregulated
ex: ungated potassium channels to keep potassium at equilibrium
47
ion channels- voltage-gated channels
regulated by the membrane potential change near the channel
48
ion channels- ligand-gated channels
regulated by the binding of a specific substance or ligand to the channel
49
enzyme-linked receptors
Membrane receptors that display catalytic activity in response to ligand binding
Have three primary protein domains
Often results in the initiation of a second messenger cascade
50
three domains of enzyme linked receptors
Membrane spanning domain- anchors the receptor in the cell membrane
Ligand-binding domain- stimulated by the appropriate ligand
Induces a conformational change that activates the catalytic domain
Catalytic domain exhibits the function, often second messenger
51
G protein-coupled receptors
Large family of integral membrane proteins involved in signal transduction
Has 7 membrane spanning alpha-helices
In order to transmit signals, they use a heterotrimeric G protein
52
heterotrimic G protein of G protein-coupled receptors
alpha, beta, and gamma subunits interaction
alpha subunit binds GDP and is in a complex with the beta and gamma subunits
Once GDP is replaced with GTP, the alpha subunit is able to dissociate from the beta and gamma subunits
the activated alpha subunit alters the activity of adenylate cyclase, either activating or inhibiting it
Once the GTP is dephosphorylated to GDP, the alpha subunit will rebind to the beta and gamma subunits
The binding of the G protein represents a switch to the active state and affects the intracellular signalling pathway
53
Three main types of G proteins
Gs stimulates adenylate cyclase - increases levels of cAMP
Gi inhibits adenylate cyclase - decreases levels of cAMP
Gq activates phospholipase C - forms PIP - increases levels of IP3
-IP3 opens calcium channels in the endoplasmic reticulum, increasing calcium levels
54
Homogenization of protein
crushing, grinding, or blending the tissue into an evenly mixed solution
55
migration velocity equation
V = Ez/f
migration velocity is proportional to to electric field strength and the net charge on the molecule, inversely proportional to the frictional coefficient f
f is dependent on the mass and shape of the molecule
56
native PAGE and limitations/benefits
Polyacrylamide gel electrophoresis
Analyzes proteins in their native states
Limited by varying size combined with varying mass-to-size ratios of cellular proteins
multiple proteins may experience the same level of migration functional
native protein can be recovered if no stain has been use
57
SDS Page and limitations/benefits
separates proteins based on mass alone
The addition of sodium dodecyl sulfate neutralizes the proteins original charge with large chain negative charges and denatures the proteins
58
Isoelectric focusing; anode/cathode
Separated on the basis of isoelectric point
mixture of proteins is placed in a gel with a pH gradient
acidic at positive anode, basic at negative cathode
59
Chromatography general principles
the more similar the compound is to its surroundings the more slowly it will move through its surroundings
Stationary phase/adsorbent + mobile phase
varying retention times of each compound in the solution results in separation of the components within the stationary phase, or partitioning
60
column chromatography
a column is filled with polar beads and a nonpolar solvent is poured through
the solvents qualities can be changed to help elute the protein of interest
the less polar the compound the faster it can elute through
can be used to separate and collect other macromolecules besides protein, such as nucleic acids
61
ion-exchange chromatography
a column is filled with charged beads to attract compounds with an opposite charge
62
size-exclusion chromatography
a column is filled with beads containing tiny pores, small compounds get caught in the pores and take longer to travel through
63
affinity chromatography
customized column to bind any protein of interest once the protein is retained in the column, it can be eluted by washing the column with a free receptor (or target or antibody), which will compete with the bead-bound receptor and free the protein from the column
64
typical methods of determining protein structure
X-ray crystallography and nuclear magnetic resonance can determine protein structure
65
determining composition of small proteins
Small proteins are best analyzed with the Edman degradation, which uses cleavage to sequence proteins of up to 50 to 70 amino acids selectively and sequentially removes the N-terminal amino acid of the protein, which can be analyzed via mass spectroscopy
66
determining composition of larger proteins
Larger proteins can use chymotrypsin, trypsin, and cyanogen bromide
67
activity analysis typical procedure
protein activity is generally determined by monitoring a known reaction with a given concentration of substrate and comparing it to a standard
68
Concentration determination- spectroscopy
Concentration is determined almost exclusively through spectroscopy
UV spectroscopy can detect aromatic side chains
This is sensitive to sample contaminates
69
Concentration determination- Bradford protein assay
one of several reactions that causes colorimetric changes in response varying levels of concentration -most popular because reliable and simple
mixes a protein with Coomassie Brilliant Blue
-The dye gives up protons in response to amino links
increased concentration causes larger concentrations of blue dye
-less accurate for multiple proteins, limited by presence of detergent or excessive buffer
70
Carbohydrate nomenclature
Aldoses are carbohydrates that contain an aldehyde group
Ketose are carbohydrates that contain a ketone group (completely oxidized)
71
Glyceraldehyde structure
simplest aldose (CH2-OH)-(CHOH)-(CHO)
72
Dihydroxyacetone
simplest ketose (CH2-OH)-(CO)-(CH2-OH)
73
Stereoisomers, enantiomers, epimers
In terms of difference in chirality:
Stereoisomers differ at more than one
enantiomers differ at all
epimers differ at only one
74
number of stereoisomers with common backbone equation
2^number of chiral centers
75
D or L assignment
All monosaccharides are assigned D or L based on whether the furthest hydroxide from the carbonyl points towards the right(D) or the left(L)
76
Fischer projection conversion
Sides of a skeleton model = Wedge = coming towards you
77
Cyclic sugar formation
Monosaccharides contain both a nucleophilic hydroxyl group and an electrophilic carbonyl group, which can allow them to form cyclic hemiacetals and hemiketals
78
nomenclature cyclic monosaccharides
hemiacetals are from aldoses
hemiketals are from ketones
pyranoses are six carbon
furanose are five carbon
79
anomer definition
anomers are epimers that differ at their anomeric carbon
80
alpha vs beta anomer
alpha anomer has the -OH group trans and down to the CH2OH substituent
beta anomer has the -OH group cis and up to the CH2OH substituent
81
Haworth and Fischer conversion
When we convert from straight chain to haworth (cyclic) projection, any group on the right in the fischer projection will point down in the Haworth projection
in determining L or D assignment, remember that D can equal down and l can equal left
82
Mutarotation
exposing hemiacetal rings to water will cause them to spontaneously cycle between the open and closed form
Because the substituents on the single bond between C-1 and C-2 can rotate freely, either the alpha or beta anomer can be formed
the alpha is less favored due to the axial hydroxyl group adding steric strain
83
Oxidized aldoses and reducing sugars
Oxidized aldoses are called aldonic acids
any monosaccharide with a hemiacetal ring is considered a reducing sugar
84
test for reducing sugars (hemiacetal rings)
Tollen’s reagent is Ag(NH3)2, tests for reducing sugars -produces a mirror like product
Benedict’s reagent is from copper, tests for reducing sugars -produces a dark red product
ketose sugars are also reducing sugars and give positive results to those tests under basic conditions, where they tautomerize to form aldoses
85
Esterification of carbohydrates
carbohydrates can participate in reactions with carboxylic acids and derivatives to form esters
86
Glycoside formation from carbohydrates
Hemiacetals react with alcohols to form acetals
The resulting carbon-oxygen bond are called glycosidic bonds, the acetals formed are glycosides
87
Disaccharide formation and three important disaccharides
Glycosidic bonds formed between hydroxyl groups of two monosaccharides form a disaccharide
Sucrose(GluFru), lactose(GluGal), and maltose(GluGlu)
88
Cellulose
beta-D-glucose molecules linked via beta-1,4 glycosidic bonds
hydrogen bonds holding the chains together for support
89
Starches
alpha-d-glucose molecules linked via alpha-1,4-glycosidic bonds (amylose) or alpha-1,4 AND alpha-1,6-glycosidic bonds (amylopectin)
Iodine tests for starch
beta-amylase cleaves at nonreducing end, alpha-amylase cleaves randomly
90
Glycogen and Glycogen phosphorylase
similar to starch but with more alpha-1,6-glycosidic bonds
-1/10 as opposed to 1/25
Glycogen phosphorylase cleaves glucose from the nonreducing end and phosphorylates it, producing glucose 1-phosphate -important for metabolism
91
Lipid saturation
saturated only have single bonds, more stable structure, form solids
unsaturated have double bonds, less stable structure, form liquids
92
phospholipids
contain a phosphate and alcohol joined to a fatty acid tail by phosphodiester linkages
can be further classified according to the backbone
93
glycerophospholipids
a type of phospholipid that contains a glycerol backbone bonded by ester linkages to two fatty acids and by a phosphodiester linkage to a highly polar head group membrane lipid
important in cell recognition, signalling, and binding
94
sphingolipids
like glycerophospholipids in that they are sites of recognition and bonded at cell surface
has a sphingosine or sphingoid backbone
many are phospholipids because they contain a phosphodiester linkage four groups
95
Four groups of sphingolipids
Sphingophospholipids
Sphingomyelins
Glycosphingolipids
Gangliosides
Sphingophospholipids contain phosphodiester bond and are therefore a type of phospholipids
Sphingomyelins are the major class of sphingophospholipids and contain a phosphatidylcholine or phosphatidylethanolamine head group
-major component of the myelin sheath
Glycosphingolipids are attached to sugar moieties instead of a phosphate group
- Cerebrosides have one sugar, globosides have more than one sugar
- Outer surface of the plasma membrane
Gangliosides contain oligosaccharides with at least one terminal
-NANA, sialic acid -Cell interaction, recognition, and signal transduction
96
waxes
esters of long-chain fatty acids with long-chain alcohols function for protection and to prevent evaporation (plants) or dehydration (animals)
97
signaling lipids general mechanisms
lipids serve as coenzymes in the electron transport chain and in glycosylation reactions
98
terpenes and terpenoids
built from isoprene (C5H8)
metabolic precursors to steroids and other lipid signaling molecules like vitamin A
varied independent functions
Terpenoids are derivatives of terpenes that have undergone oxygenation or rearrangement of the carbon skeleton
Further modified (like terpenes) by the addition of an extensive variety of functional groups
99
terpene/terpenoid nomenclature
Group according to the number of isoprene units
a single terpene unit contains two isoprene units
Monoterpenes (C10H16) are two isoprene units
Sesquiterpenes are three isoprene units
Diterpenes are four isoprene units…
100
steroids and cholesterol
metabolic derivatives of terpenes, nonpolar
steroid hormones are steroids that act as hormones
testosterone, estrogens, cortisol, aldosterone
Cholesterol
- steroid and steroid precursor
- major component of the phospholipid bilayer responsible for membrane fluidity
101
prostaglandins
produced by all cells, not just prostate gland cells
unsaturated carboxylic acids derived from arachidonic acid
regulate the synthesis of cAMP, which in turn regulates other hormones
- downstream effects include effects on smooth muscle, sleep-wake cycle, elevation of body temperature
- NSAIDs like aspirin inhibit the production COX, part of the prostaglandins pathway
102
which are the fat-soluble vitamins
ADEK
103
vitamin A
vitamin A - carotene unsaturated hydrocarbon important in vision, growth and development, and immune function aldehyde form retinal
- eyesight carboxylic acid form retinoic acid
- regulates gene expression during epithelial development
104
vitamin D
vitamin D
- cholecalciferol can be consumed or formed from UV light in the skin
converted to calcitriol in kidneys (biologically active)
increases calcium and phosphate uptake, which promotes bone growth
Rickets if lack of vitamin D
105
vitamin E
substituted aromatic ring with a long isoprenoid reacts with free radicals (biological antioxidant)
106
vitamin K
a group of compounds including phylloquinone and the menaquinones vital to the production of prothrombin (clotting factor)
also introduces calcium-binding sites on several calcium-dependent proteins
107
triacylglycerols
most efficient way to store energy, twice as good as carbohydrates
three fatty acids bonded by ester linkages to glycerol
adipocytes store large amount of triacylglycerols
physical characteristics primarily determined by the level of saturation primarily observed as oily droplets in cytosol
108
Free fatty acids and saponification
Free fatty acids are unesterified fatty acids with a free carboxylate group
Saponification is the ester hydrolysis of triacylglycerols using a strong base known as lye creates soaps which act as surfactants (lower surface tension)
109
Nucleosides vs nucleotides
Nucleosides are a pentose bonded to a nitrogenous base with a covalent linkage to C-1’ of the sugar
Nucleotides are formed when one or more phosphate groups are attached to the C-5’ of the sugar
110
sugar-phosphate backbone
the backbone of DNA is alternating sugar and phosphate group
111
purines and pyrimidines, identiy each one
Purines are Adenine (-NH2) and Guanine (=O) two rings
Pyrimidines are Cytosine (-NH2), Uracil (=O x2), and Thymine(-CH3) examples of biological aromatic heterocycles
112
B-DNA and Z-DNA
The double helix of most DNA is a right-handed helix (B-DNA) makes a turn every 3.4nm and contains about 10 bases within that span
left-handed Z-DNA has a zigzag appearance has a turn every 4.6nm and contains about 12 bases within each turn high GC content or high salt concentration
113
Denaturation of protein
Heat, alkaline pH, formaldehyde and urea can denature DNA
DNA can be annealed if the denaturing condition is slowly removed important step in PCR
114
Eukaryotic chromosome organization - Histones
DNA that makes up a chromosome is wound around a group of small basic proteins called histones, forming chromatin
There are five histone proteins, two copies each form a histone core
about 200 base pairs of DNA wrap around this complex, forming a nucleosome examples of nucleoproteins (proteins that associate with DNA)
115
Eukaryotic chromosome organization- Heterochromatin and Euchromatin
During interphase chromosomes have a diffuse configuration, chromatin
A small percentage of the chromatin remains compacted during interphase and is referred to as heterochromatin, whereas the rest is called euchromatin
Heterochromatin - appears dark under light, is transcriptionally silent
Euchromatin - appears light under light, is genetically active
116
Eukaryotic chromosome organization- Telomeres and Centromeres
Telomeres are maintained by telomerase, which is more highly expressed in rapidly dividing cells
have a high GC content
Centromeres are regions of DNA found in the center of chromosomes, composed of heterochromatin, and also with high GC content
During cell division, the two sister chromatids can therefore remain connected at the centromere until microtubules separate the chromatids during anaphase
117
DNA replication- origin
The replisome or replication complex is a set of specialized proteins that assist the DNA polymerases
DNA begins replication at the origin of replication
-In bacterial chromosomes there is a single origin of replication on a closed, double-stranded circular DNA molecule
As the replication forks move toward each other and sister chromatids are created, the chromatids will remain connected at the centromere
Helicase is responsible for unwinding the DNA ssDNA-binding proteins bind to the unraveled strand, preventing both the reassociation of the DNA strands and the degradation of DNA by nucleases
DNA topoisomerases reduce the torsion of supercoiling be introducing negative supercoils
118
DNA replication- Synthesis of daughter strands
primase synthesizes a short RNA primer to start replication on each strand
DNA polymerase III (prokaryotes) or DNA polymerases alpha, beta, and epsilon begin synthesizing the daughter strands of DNA in the 5’ to 3’ direction
-DNA polymerases read the template in a 3’ to 5’ direction
DNA polymerase I (prokaryotes) or RNase H (eukaryotes) removes the RNA primer
DNA polymerase I (prokaryotes) or DNA polymerase gamma (eukaryotes) adds DNA nucleotides to where the primer had been
DNA ligase seals the ends of the DNA molecules together
119
DNA replication- Replicating the ends
DNA polymerase cannot complete synthesis of the 5’ end so it keeps getting shorter -telomeres help protect against this
120
DNA repair- oncogenes and tumor suppressor genes
mutated genes that cause cancer are termed oncogenes
-before these genes mutate, they are referred to as proto-oncogenes
tumor suppressor genes, like p53 or Rb (retinoblastoma), encode proteins that inhibit the cell cycle or participate in DNA repair processes
-sometimes called antioncogenes
121
DNA proofreading and mismatch repair
proofreading
- part of the DNA polymerase enzyme proofreads the enzyme
- looks at the level of methylation to determine which one needs to be repaired, the template strand will have higher methylation
- DNA ligase lacks proofreading ability
- the lagging strand is much more likely to have mutations
mismatch repair
- G2 phase cells have machinary for mismatch repair
- detect and remove errors introduce in replication that were missed during S phase encoded by genes MSH2 and MLH1
122
nucleotide excision repair
UV light induces the formation of dimers between adjacent thymine residues in DNA, which distorts the shape of the double helix.
These are eliminated by a nucleotide excision repair (NER)
- cut and patch process, specific proteins scan the DNA molecule and recognize the lesion as a bulge
- an excision endonuclease makes nicks in the phosphodiester backbone of the damaged strand on both sides of the thymine dimer and removes the defective oligonucleotide
123
base excision repair
the affected base is recognized and removed by a glycosylase enzyme, leaving behind an apurinic/apyrimidinic (AP) site, or abasic site
this site is recognized by an AP endonuclease that removes the damaged sequence from the DNA
DNA polymerase and DNA ligase can then fill in the gap and seal the strand
124
recombinant DNA biotechnology purpose
Recombinant DNA technology allows a DNA fragment from any source to be multiplied by either gene cloning or polymerase chain reaction
125
DNA cloning steps
1 Cloning requires the investigator ligate the DNA of interest into a piece of nucleic acid referred to as a vector, forming a recombinant vector Vectors are usually bacterial or viral plasmids
2 The vector is then transferred to a host bacterium which is grown in colonies, and a colony containing the recombinant vector is isolated (accomplished because the recombinant vector also contains a gene for antibiotic resistance)
3 The bacteria can then be made to express the gene of interest to gather protein, or be lysed to reisolate the replicated recombinant vectors
126
Restriction enzymes
Endonucleases that recognize specific palindromic double-stranded DNA sequences
- Isolated from bacteria
- some produce offset cuts, yielding sticky ends that are advantageous in facilitating the recombination of a restriction fragment with the vector DNA
127
DNA libraries and cDNA
DNA fragments are cloned into vectors and can be utilized for further study
cDNA (complementary DNA) libraries are constructed by reverse-transcribing processed mRNA
-cDNA lacks introns, these libraries are sometimes called expression libraries
128
hybridization definition
the joining of complementary base pair sequences
129
polymerase chain reaction (PCR)
1 Know the sequences that flank the desired region of DNA
2 Use primers with high GC content that are complementary to the DNA that flanks the desired region
3 DNA polymerase from an extremophile bacteria is used to replicate due to hot temperatures
4 The DNA of interest is denatured, replicated, and then cooled to allow reannealing of the daughter strands with the parent strands. This process is repeated several times, doubling the amount of DNA with each cycle, until enough copies are available
130
Gel electrophoresis
all DNA strands will migrate toward the anode of an electrochemical cell
the longer the strand the slower it will migrate
131
Southern blot
A southern blot detects the presence and quantity of various DNA strands by using radiolabeled or indicator probes made from complementary DNA
-DNA is cut by restriction enzymes beforehand
132
DNA sequencing
uses template DNA, primers, DNA polymerase, all four dNTPs, and ddNTPs as well
- the ddNTPs contain a hydrogen rather than a hydroxyl group, once they are added the chain cannot extend the fragments are separated by size using gel electrophoresis, and the last base for each fragment can be read
- because it separates by size, the bases can be read in order
133
Gene therapy
transfers a normal copy of the gene efficient gene delivery vectors must be used, most are modified viruses
134
Transgenic and Knockout Mice
transgenic mice are altered at their germ line through the introduction of a cloned gene, referred to as a transgene
knockout mice are transgenic mice with a gene removed instead of introduced
serve as models
135
mRNA purpose
carries the information specifying the amino acid sequence to the ribosome in eukaryotes
monocistronic (only codes for one protein product), in prokaryotes can be polycistronic
-starting the process of translation at different locations in the mRNA can result in different proteins in prokaryotes
136
tRNA purpose
responsible for converting the language of nucleic acids into the language of amino acids/proteins
Each type of amino acid requires two high-energy bonds from ATP, the attachment of the amino acid is an energy-rich bond
The aminoacyl-tRNA synthetase transfers the activated amino acid to the 3’ end of the correct tRNA
Each tRNA has a CCA nucleotide sequence where the amino acid binds
137
rRNA purpose
synthesized in the nucleolus for the ribosomal machinery
many rRNA molecules function as ribozymes (enzymes made of RNA molecules)
138
Codons, stop codons
three letter gene sequence recognized by anticodons to translate into an amino acid
Stop codons are UGA, UAG, UAA
139
Missense, nonsense, and frameshift mutations
Missense one amino acid substitutes for another
Nonsense premature stop codon
Frameshift insertions or deletions shift the reading frame
140
Transcription location and why
the machinery to generate a protein is located in the cytoplasm
DNA cannot leave the nucleus, so it must use RNA to transmit genetic information
141
Mechanism of transcription
helicase and topoisomerase unwind the double-stranded DNA
RNA polymerase locates genes by searching for specialized DNA regions known as promoter regions
RNA polymerase II binds to the TATA box of the promoter region
- transcription factors help the RNA polymerase locate and bind
- TATA box is -25 nucleotides upstream travels in a 3’ -\> 5’ direction does not proofread
142
Types of Eukaryotic RNA polymerase
RNA Polymerase I - synthesizes rRNA
RNA Polymerase II - synthesizes hnRNA (pre-processed mRNA)
RNA Polymerase III - synthesizes tRNA and some rRNA
143
types of posttranscriptional processing in eukaryotes
splicing, 5’ Cap, 3’ Poly-A tail
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splicing
removing introns and ligating exons using the spliceosome, where snRNA/snRNPs complex recognize both the 5’ and 3’ ends of the introns, and they are excised in the form of a lariat
145
5' cap
a 7-methylguanylate triphosphate cap added to the 5’ end protects the mRNA from degradation
146
3' Poly-A tail
a polyadenosyl tail is added to the 3’ end of the mRNA transcript to protect against degradation
the longer the tail the more time the mRNA will be able to survive before being digested in the cytoplasm
147
alternative processing
the primary transcript of hnRNA may be spliced together in different ways to produce multiple variants of protein
148
Ribosome relationship to RNA
has three binding sites for tRNA: A site, P site, E site
has one binding site for mRNA,
once the mRNA binds the two subunits come together, creating a compact that keeps the mRNA and tRNA in stable and proper orientation for protein synthesis
149
prokaryotic vs eukaryotic ribosomes
Eukaryotic has a 40S and a 60S subunit making an 80S ribosome
Prokaryotic has a 30S and a 50S subunit making a 70S ribosome
150
timing of translation prokaryotes vs eukaryotes
In prokaryotes, the ribosomes start translating before the mRNA is complete
In eukaryotes, transcription and translation occur at separate times and in separate locations
151
Initiation of translation
In prokaryotes, the small subunit binds to the Shine-Dalgarno sequence in the 5’ untranslated region of the mRNA
In eukaryotes, the small subunit binds to the 5’ cap structure
The charged initiator tRNA binds to the AUG start codon
The large subunit then binds to the small subunit
-assisted by initiation factors
152
Elongation of translation
The A site receives the incoming amino acid
The P site holds the tRNA that carries the growing chain
- also where the first amino acid (met) binds
- A peptide bond is formed between the P and A site AA
--this requires peptidyl transferase, GTP is used
The E site is where the now uncharged tRNA unbinds
Elongation factors assist by locating and recruiting aminoacyl-tRNA along with GTP
Signal sequences in some eukaryotic proteins designate a particular destination for the protein
153
Termination of translation
When any of the three stop codons moves into the A site, a release factor binds to the termination codon, causing hydrolysis of the completed polypeptide chain from the final tRNA
154
Posttranslational processing
Chaperones assist in the protein-folding process after synthesis
Many proteins are modified by cleavage events
Other biomolecules may be added to the peptide
155
covalent additions to peptide in posttranslation
Phosphorylation - addition of phosphate group
Carboxylation - addition of carboxylic acid group
Glycosylation - addition of oligosaccharides
Prenylation - addition of lipid groups
156
Prokaryotic translation- operons definition
“On-off switch” in which genes share a promoter and are transcribed as a group
157
Jacob-Monod model
Jacob-Monod model describes the structure and function of operons
structural genes - codes for proteins of interest
operator site - capable of binding a repressor protein
promoter site - similar to eukaryotic promoters, RNA polymerase binds
a regulator gene - codes for a protein known as the repressor
158
Inducible systems
the repressor is bonded tightly, this system is negative control
to remove the block an inducer must bind the repressor protein
operate similar to competitive inhibition, as the concentration of inducer increases it pulls more of the repressor off of the operator region, freeing up those genes for transcription
159
lac operon
inducible system bacteria can digest lactose but prefer glucose
the lac operon only operates in the presence of lactose
assisted by binding of catabolite activator protein (CAP)
example of positive control
160
Repressible Systems
allow for constant production, the repressor is inactive until it binds to a corepressor
tends to serve as negative feedback
161
trp operon
tryptophan acts as a corepressor, the cell turns off its machinery to synthesize its own tryptophan
162
eukaryotes- transcription factors
recognizes specific sequences and recruits transcriptional machinery
DNA binding domain - recognizes certain sites and binds
Activation domain - allows for the binding of several transcription factors and important regulatory proteins
163
Enhancers
Response elements can be recognized by specific transcription factors, several response elements may be grouped together to form an enhancer, which allows for the control of one gene’s expression by multiple signals
Signal molecules such as cyclic AMP, cortisol, and estrogen bind to specific receptors
Enhancer regions can be up to 1000 bp away from the gene they regulate, even located within an intron
-in contrast, upstream promoter elements must be within 25 bases
164
Gene duplication
genes can be duplicated in series on the same chromosome
genes can also be duplicated in parallel by opening the gene with helicases and permitting DNA replication only of that gene
165
Regulation of Chromatin Structure - Histone Acetylation
histone acetylases are recruited by transcription factors
these acetylate lysine residues of histone proteins, decreasing the positive charge on lysine residues and weakening the interaction of the histone with DNA, resulting in an open chromatin conformation
histone deacetylases do the opposite
166
DNA Methylation
DNA methylases add methyl groups to cytosine and adenine nucleotides linked to gene silencing
- important in development, where it silences genes that no longer need to be activated
- heterochromatin is much more heavily methylated
167
general membrane structure and function
fat-soluble compounds cross easily, while larger and water-soluble compounds need alternative entry
Carbohydrates associated with membrane-bound proteins create a glycoprotein coat
Proteins embedded act as cellular receptors during signal transduction
168
Membrane dynamics in the fluid mosaic model, lipid rafts, flippases
Phospholipids move rapidly in the plane of the membrane through simple diffusion
lipid rafts are collections of similar lipids (maybe with associated proteins) that serve as attachment points for other biomolecules and play a role in signalling
lipid rafts and proteins travel more slowly through the membrane
flippases assist in the transition or “flip” between layers
Many cells can up- or downregulate the number of specific cellular receptors on their surface in order to meet cellular requirements
169
Membrane components - Fatty Acids and Triacylglycerols
when incorporated into phospholipid membranes, saturated fatty acids decrease the overall membrane fluidity
170
Membrane components - Phospholipids
substituting one of the fatty acid chains of triacylglycerol with a phosphate group
a polar group joins the nonpolar tails
spontaneously assemble into micelles or liposomes
serve structural roles and can act as second messengers
171
Membrane components - Sphingolipids
similar to phospholipids but without a glycerol
have various classes depending on the identity of their hydrophilic regions
172
Membrane components - Cholesterol and Steroids
cholesterol regulates membrane fluidity
at lower temperatures, cholesterol occupies the space between adjacent phospholipids prevents crystal structure formation
at higher temperatures it limits movement of phospholipids within the bilayer
by mass, it composes 20%; by mole fraction, 50%
173
Membrane components - Waxes
long chain fatty acid and long chain alcohol, rarely present in cell membrane unless in plants
can provide both stability and rigidity within the nonpolar tail region only
174
Membrane components - Proteins
together, transmembrane (through) and embedded (interior) proteins are considered integral because they associate with the interior of the membrane
membrane-associated (peripheral) proteins are bound to the lipid bilayer
175
Membrane components - Carbohydrates
generally attached to protein molecules on the extracellular surface, act as signaling and recognition molecules
blood antigens are sphingolipids that differ only in carbohydrate sequence
176
Membrane receptors
can activate or deactivate transporter for facilitated diffusion
tend to be transmembrane proteins
- ligand-gated ion channels
- g-protein coupled receptors
177
Gap Junctions/Connexons
allow for direct cell-cell communication
permit movement of water and some solutes (not proteins)
formed by the alignment and interaction of pores composed of six molecules of connexin
178
Tight Junctions
prevent solutes from leaking into the space between cells via a paracellular route form a continuous band around the cell
179
Desmosomes
bind adjacent cells by anchoring to their cytoskeletons
formed by interactions between transmembrane proteins
associated with intermediate filaments
primarily found at the interface between two layers of epithelial tissue
hemidesmosomes attach epithelial cells to underlying structures
180
Concentration Gradients
determine whether active or passive transport
181
Simple diffusion
most basic, substrates move down their concentration gradient directly across the membrane
182
Osmosis
simple diffusion of water driven by osmotic pressure, a colligative property
-dependent only on concentration
183
Osmotic pressure equation
Osmotic Pressure (∏) = iMRT
M is molarity, R is ideal gas constant, T is the absolute temperature, i is the van’t Hoff factor
thought of as a “sucking” pressure
184
Facilitated diffusion
simple diffusion for molecules that are impermeable to the membrane using proteins uses carriers and channels
185
Facilitated diffusion - carriers
open to only one side, like a revolving door in their function
binding of the substrate molecule induces the occluded state, where neither side is open
186
Facilitated diffusion - channels
may be in open or closed conformation, in open conformation both sides are exposed much more rapid transport kinetics
187
Active transport, primary vs secondary
primary active transport
-uses ATP or another energy molecule
secondary active transport -“coupled transport”
- harnesses the energy released by one particle going down its electrochemical gradient to drive a different particle up
- Symport- both particles same direction
- Antiport- particles flow in opposite directions
188
Endocytosis
pinocytosis- the endocytosis of fluids and dissolved particles
phagocytosis- the ingestion of large solids like bacteria
189
Exocytosis
secretory vesicles fuse with the membrane, releasing their contents to the environment
important in the nervous system and intracellular signalling neurotransmitters from synaptic vesicles
190
Nernst equation
used to determine the membrane potential from the intra- and extracellular concentrations of the various ions
E = 61.5/(charge of ion) \* log([ion]outside/[ion]inside)
191
Sodium potassium pump
Cell membranes are more permeable to K+ ions than Na+ ions at rest due to more K+ channels
Na+/K+ channels maintain the gradient
192
outer mitochondrial membrane special characteristic
highly permeable
193
inner mitochondrial membrane special characteristics
more restricted permeability
contains numerous infoldings known as cristae which increase surface area
encloses the mitochondrial matrix
-citric acid cycle high level of cardiolipin, no cholesterol
194
GLUT 2 (location, Km)
hepatocytes and pancreatic cells the Km is quite high, the liver will pick up glucose in proportion to its concentration in the blood (first-order kinetics)
195
GLUT 2 (purpose)
captures excess glucose after a meal for storage
When the glucose concentration drops below the Km for the transporter, much of the remainder bypasses the liver and enters the peripheral circulation
the liver will pick up excess glucose and store it preferentially after a meal, when blood glucose levels are high
In the beta-islet cells of the pancreas, GLUT 2 along with glucokinase serves as the glucose sensor
196
GLUT 4 (location, Km)
adipose tissue and muscle
the Km is close to normal blood glucose levels, so the transporter is saturated when blood glucose levels are slightly above normal
197
GLUT 4 (purpose)
responds to the glucose concentrations in peripheral blood
when a person has high blood sugar the transporter still maintains only a constant rate of glucose influx (first-order kinetics)
Muscle and adipose tissue requires more glucose
Muscle stores excess glucose as glycogen
adipose tissue requires glucose to form dihydroxyacetone phosphate (DHAP), which is converted to glycerol phosphate to store incoming fatty acids as triacylglycerol
198
Glycolysis and order
Glucose Glucose-6-P Fructose-6-P Fructose-1,6-bP Glyceraldehyde-3-phosphate 1,3-Biphosphoglycerate 3-Phosphoglycerate 2-Phosphoglycerate Phosphoenolpyruvate Pyruvate
Cytoplasmic pathway that converts glucose into 2 pyruvate molecules, releasing a modest amount of energy captured in two substrate level phosphorylations and one oxidation reaction
All cells undergo glycolysis
Provides intermediates for other pathways
199
Important enzymes of glycolysis, first step
ATP or NADH?
Hexokinase and glucokinase
the first steps of glycolysis, phosphorylation of glucose to glucose-6-P to prevent glucose from leaving via the transporter
-glucose enters the cell by facilitated diffusion or active transport
Hexokinase is widely distributed and inhibited by its product, G-6-P
Glucokinase is found only in liver cells and pancreatic beta-islet cells, and is induced by insulin in the liver
200
Important enzymes of glycolysis, fructose 6-phosphate -\> ?
ATP or NADH?
Phosphofructokinase-1
**rate-limiting enzyme of glycolysis**
phosphorylates fructose 6-phosphate to fructose 1,6-phosphate
inhibited by ATP and citrate, and activated by AMP
insulin stimulates and glucagon inhibits PFK-1 -through the PFK-2 mechanism, which activates F2,6-bp, which activates PFK-1
201
Important enzymes of glycolysis, glyceraldehyde-3-P -\> ?
ATP or NADH?
Glyceraldehyde-3-Phosphate dehydrogenase
Glyceraldehyde-3-Phosphate -\> 1,3-biphosphoglycerate and reduction of NAD+ to NADH
If glycolysis is aerobic, the NADH can be oxidized by the mitochondrial matrix
202
Important enzymes of glycolysis, 1,3-biphosphoglycerate -\> ?
ATP or NADH?
3-Phosphoglycerate Kinase transfers the high-energy phosphate from 1,3-bisphosphoglycerate to ADP, forming ATP and 3-phosphoglycerate
203
Important enzymes of glycolysis, Phosphoenolpyruvate -\> ?
ATP or NADH?
Pyruvate Kinase
Phosphorylation of ADP to ATP and phosphoenolpyruvate to pyruvate
activated by fructose 1,6-bisphosphate feed-forward activation
204
Irreversible enzymes of glycolysis
Glucokinase/Hexokinase
PFK-1
Pyruvate Kinase
205
Fermentation; key enzyme and purpose
Occurs in the absence of oxygen
key enzyme is lactate dehydrogenase
reduces pyruvate to lactate and oxidizes NADH to NAD+`
206
Dihydroxyacetone phosphate
an important intermediate of glycolysis used in hepatic and adipose tissue for triacylglycerol synthesis
formed from fructose 1,6-bisphosphate
207
1,3-biphosphoglycerate and PEP uses
important intermediate of glycolysis
1,3-biphosphoglycerate (1,3-BPG) and phosphoenolpyruvate (PEP) are high-energy intermediates used to generate ATP by substrate-level phosphorylation
only ATP in anaerobic respiration
208
Glycolysis in erythrocytes
anaerobic glycolysis is the only pathway for ATP production
have bisphosphoglycerate mutase, 2,3-BPG-\> 1,3-BPG
209
Galactose metabolism
Galactose comes from dietary lactose
Phosphorylated by galactokinase, trapping in the cell
Galactose-1-phosphate uridyltransferase converts galactose 1-P to glucose 1-P
210
Fructose metabolism
fructose is absorbed into the hepatic portal vein, the liver phosphorylates fructose using fructokinase to trap in the cell
aldolase B converts fructose 1-phosphate into glyceraldehyde
211
Pyruvate dehydrogenase
Pyruvate from aerobic glycolysis enters mitochondria, where pyruvate dehydrogenase converts it into acetyl-CoA
irreversible
activated in the liver by insulin
requires thiamine, NAD+
212
Three possible fates of pyruvate
conversion to acetyl-CoA by PDH
conversion to lactate by lactate dehydrogenase
conversion to oxaloacetate by pyruvate carboxylase
213
Glycogen storage location
Glycogen is stored in the cytoplasm as granules
Glycogen stored in the liver is a source of glucose between meals
214
Glycogenesis and enzymes
synthesis of glycogen granules, begins with a core protein called glycogenin
Glycogen synthase and branching enzyme
215
Glycogen synthase
the rate-limiting enzyme of glycogen synthesis
forms an alpha-1,4-glycosidic bond found in the linear glucose chains of the granule
stimulated by insulin
216
Branching enzyme
responsible for introducing alpha-1,6-linked branches into the granule as it grows
- hydrolyzes one of the alpha-1,4 bonds to release a block of oligoglucose which is then moved and added in a slightly different location
- forms an alpha-1,6 bond to create a branch
217
Glycogenolysis and enzymes
reverse of glycogenesis, degradation of glycogen to glucose
glycogen phosphorylase and debranching enzyme
218
Glycogen phosphorylase
the rate limiting enzyme of glycogenolysis
breaks an alpha-1,4 glycosidic bond
activated by glucagon in the liver, AMP and epinephrine in skeletal muscle
219
Debranching enzyme
deconstructs the branches introduce by branching enzyme
220
Gluconeogenesis, important substrates
Glycerol-3-Phosphate
Lactate
Glucogenic amino acids (from muscle proteins)
221
Important enzymes of gluconeogenesis
Most steps in gluconeogenesis represent a reversal of glycolysis, the four enzymes to know are those required to circumvent the irreversible steps of glycolysis in the liver
Pyruvate carboxylase (Pyruvate -\> OAA)
Phosphoenolpyruvate carboxykinase (PEPCK) (OAA -\> PEP)
Fructose-1,6-bisphosphatase (f 1,6 bp -\> f-6-P)
Glucose-6-phophatase
222
Pyruvate carboxylase
Activated by acetyl-Coa
pyruvate can go through pyruvate dehydrogenase as well, but that is only if the cell needs acetyl-Coa
the source is fatty acids, to produce glucose fatty acids need to be burned
**Pyruvate -\> OAA**
OAA cannot leave the the mitochondrion so it is temporarily reduced to malate to leave
223
phosphoenolpyruvate carboxykinase (PEPCK)
induced by glucagon and cortisol converts OAA to phosphoenolpyruvate (PEP) in a reaction that requires GTP
PEP goes on to produce fructose-1,6-bP
combination of pyruvate carboxylase and PEPCK circumvent the action of pyruvate kinase by converting pyruvate back into PEP
224
fructose-1,6-bisphosphatase
activated by ATP and inhibited by AMP and fructose 2,6-bisphosphate
removes phosphate from fructose 1,6-bisphosphate to produce fructose 6-phosphate
reverses the action of phosphofructokinase-1, the rate limiting step of glycolysis
225
glucose-6-phosphatase, absence in skeletal muscle
found only in the lumen of the endoplasmic reticulum in liver cells
Glucose-6-phosphate is transported into the ER, and free glucose is transported back into the cytoplasm, from where it can diffuse out of the cell using GLUT transporters
The absence of glucose-6-phosphatase in skeletal muscle means that muscle glycogen cannot serve as a source of blood glucose and rather is for use only within the muscle
Glucose -6-phosphatase is used to circumvent glucokinase and hexokinase, which convert glucose to glucose 6-phosphate
226
fructose-2,6-biphosphate
thought of as a marker for satisfactory energy levels in liver cells and controls both gluconeogenesis and glycolysis produced by PFK-2
227
Pentose phosphate pathway / Hexose monophosphate shunt and phases
occurs in the cytoplasm
Produces NADPH and serves as a source of ribose 5-phosphate for nucleotide synthesis
1st oxidative phase: rate limiting enzyme glucose-6-phosphate dehydrogenase glucose 6-phosphate to ribulose 5-phosphate produces NADPH induced by insulin
2nd nonoxidative phase: ribulose 5-phosphate to ribose-5-phosphate could be used for glycolysis or for nucleotide synthesis
228
Functions of NADPH
potent reducing agent (NAD+ is oxidizing agent)
biosynthesis of fatty acids or cholesterol
assisting in cellular bleach production in WBCs
natural antioxidant
-H2O2 is a byproduct in aerobic metabolism that can break apart to form harmful hydroxide radicals
229
Methods of forming Acetyl-CoA
Pyruvate dehydrogenase
Dihydrolipoyl transacetylase
Dihydrolipoyl dehydrogenase
Fatty acid oxidation
230
Pyruvate dehydrogenase and requirements
pyruvate is oxidized, yielding CO2 while the remaining two-carbon molecule binds covalently to TPP (vitB1).
starts the process towards production of pyruvate
Mg2+ is also required
231
Dihydrolipoyl transacetylase
The two-carbon molecule bonded to TPP is oxidized and transferred to lipoic acid, a coenzyme that is covalently bonded to the enzyme
lipoic acid’s disulfide group acts as an oxidizing agent, creating the acetyl group.
The acetyl group is now bonded to lipoic acid via thioester linkage
After this, dihydrolipoyl transacetylase catalyzes the CoA-SH interaction with the newly formed thioester link, causing transfer of an acetyl group to form acetyl-CoA
232
Dihydrolipoyl dehydrogenase
Flavin adenine dinucleotide (FAD) is used as a coenzyme in order to reoxidize lipoic acid, allowing lipoic acid to facilitate acetyl-CoA formation in future reactions
As lipoic acid is reoxidized, FAD is reduced to FADH2
In subsequent reactions, this FADH2 is reoxidized to FAD, while NAD+ is reduced to NADH
233
Fatty acid oxidation
In the cytosol, a process called activation causes a thioester bond to form between carboxyl groups of fatty acids and CoA-SH
The fatty acyl group is transferred to carnitine, whose function is merely to carry the acyl group from a cytosolic CoA-SH to a mitochondrial CoA-SH
Once acyl-CoA is formed in the matrix, beta-oxidation can occur, which removes two-carbon fragments from the carboxyl end
234
Amino Acid catabolism
Certain amino acids can be used to form acetyl-CoA.
These amino acids lose their amino group via transamination; their carbon skeletons can then form ketone bonds
235
Ketones -\> acetyl-CoA
Although acetyl-CoA is typically used to produce ketones when the pyruvate dehydrogenase complex is inhibited, the reverse reaction can occur as well
236
Alcohol -\> acetyl-CoA
The enzymes alcohol dehydrogenase and acetaldehyde dehydrogenase convert it to acetyl-CoA
This reaction is accompanied by NADH buildup, which inhibits the Krebs cycle
-the acetyl-CoA formed through this process is used primarily to synthesize fatty acids
237
Citric acid cycle general details
takes place in the mitochondrial matrix and begins with the coupling of a molecule of acetyl-Coa the a molecule of oxaloacetate
GTP and energy carriers (NADH and FADH2) are produced will not occur anaerobically
NADH and FADH2 will accumulate if oxygen is not available for the electron transport chain and will inhibit the cycle
238
Citric acid cycle order
Can I keep selling sex for money officer
Citrate isocitrate alpha-ketoglutarate succinyl-coa succinate fumarate malate oxaloacetate
239
Citric Acid cycle- Step 1 - CITRATE FORMATION
Acetyl-CoA and oxaloacetate undergo a condensation reaction to form citryl-COA, an intermediate
Hydrolysis of citryl-CoA yields citrate and CoA-SH
catalyzed by _citrate synthase_
240
Citric Acid cycle- Step 2 - CITRATE isomerized to ISOCITRATE
Citrate is isomerized to isocitrate
Water is lost from citrate, yielding cis-aconitate.
Water is added back to form isocitrate
uses the enzyme _aconitase_, requires Fe2+ results in a switching of a hydrogen and a hydroxyl group
241
Citric Acid cycle- Step 3 - alpha-KETOGLUTARATE and CO2 formation
Isocitrate is first oxidized to oxalosuccinate by _isocitrate dehydrogenase_
**rate limiting enzyme**
Oxalosuccinate is decarboxylate to produce alpha-ketoglutarate and CO2
First NADH produced
242
Citric Acid cycle- Step 4 - SUCCINYL-COA and CO2 formation
Carried out by the _alpha-ketoglutarate dehydrogenase complex_, similar in mechanism to the pyruvate dehydrogenase complex
alpha-ketoglutarate and CoA come together and produce a molecule of carbon dioxide
This carbon dioxide represents the second and last carbon lost from the cycle
_Another NADH is produced_
243
Citric Acid cycle- Step 5 - SUCCINATE formation
Hydrolysis of the thioester bond on succinyl-CoA yields succinate and CoA-SH, and is coupled to the phosphorylation of GDP to GTP
catalyzed by _succinyl-CoA synthetase_
-synthetases, unlike synthases, create new covalent bonds with energy input
244
Citric Acid cycle- Step 6 - FUMARATE formation
Takes place on the inner membrane, not the mitochondrial matrix
Succinate undergoes oxidation to yield fumarate catalyzed by _succinate dehydrogenase_
- considered a flavoprotein because it is covalently bonded to FAD
- integral protein on the inner mitochondrial membrane
As succinate is oxidized to fumarate, _FAD is reduced to FADH2_
--FAD is the electron acceptor in this reaction because the reducing power of succinate is not great enough to reduce NAD+
245
Citric Acid cycle- Step 7 - MALATE formation
The enzyme _fumarase_ catalyzes the hydrolysis of the alkene bond in fumarate, yielding malate
246
Citric Acid cycle- Step 8 - OXALOACETATE formed anew
_malate dehydrogenase_ catalyzes the oxidation of malate to oxaloacetate
a third and final molecule of _NAD+ is reduced to NADH_
247
Net Results and ATP Yield of pyruvate dehydrogenase complex, citric acid cycle, glycolysis
Pyruvate dehydrogenase complex
Pyruvate + CoA-SH + NAD+ =\> acetyl-CoA + NADH + CO2 + H+
Citric Acid Cycle
Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2H2O =\> 2 CO2 + CoA-SH + 3 NADH + 3H+ + FADH2 + GTP ATP
Production 4 NADH + 1 FADH2 + 1 GTP = 25ATP per glucose + glycolysis = 30-32 ATP
248
Pyruvate dehydrogenase complex modification/regulation
can be phosphorylated by pyruvate dehydrogenase kinase
-deactivates in response to high ATP
can be dephosphorylated by pyruvate dehydrogenase phosphatase
-activates in response to high ADP
249
Takeaway major control of citric acid cycle
the ATP/ADP ratio and NADH/NAD+ ratio determine inhibition/activation
future products (succinylcholine CoA, citrate for citrate synthase) inhibit enzymes
250
Electron transport chain electron flow and complexes
Complex I - NADH dehydrogenase transfers electrons from NADH to coenzyme Q
Complex II - Succinate dehydrogenase transfers electrons from FADH2 to coenzyme Q
Ubiquinone (Coenzyme Q) - transfers electrons from complex I and II to complex III
Complex III - Cytochrome C reductase enzyme, carriers electrons to to cytochrome C
Cytochrome C - transfers electrons from complex 3 to complex 4
Complex IV - Cytochrome C oxidase enzyme, converts oxygen to water, pumps protons into intermembrane space, creating the proton motive force
251
NADH shuttles purpose
NADH formed through glycolysis cannot directly cross into the mitochondrial matrix
252
Glycerol 3-Phosphate shuttle
one isoform of glycerol-3-phosphate dehydrogenase, which oxidizes cytosolic NADH to NAD+
-FAD is then reduced on the other side to FADH2, which transfers its electrons to the ETC via complex II
253
Malate-aspartate shuttle
Cytosolic oxaloacetate is reduced to malate by malate dehydrogenase, accompanied by the oxidation of cytosolic NADH to NAD+
254
Chemiosmotic coupling, ATP synthase structure
ATP synthase contains a F0 portion and a F1 portion
The F0 portion functions as an ion channel, allowing protons to travel back along their gradient
The F1 portion utilizes the energy released from this electrochemical gradient to phosphorylate ADP to ATP
-hypothesized to work as a turbine
255
Regulation of oxidative phosphorylation
ADP and O2 are the key regulators of oxidative phosphorylation.
If O2 is limited, the rate of oxidative phosphorylation decreases, and the concentrations of NADH and FADH2 increase. The accumulation of NADH inhibits the citric acid cycle.
-respiratory control
ADP accumulation signals the need for ATP synthesis.
ADP allosterically activates isocitrate dehydrogenase, thereby increasing the rate of the citric acid cycle and the production of NADH and FADH2
256
Lipid digestion
in the duodenum and rest of small intestine pancreas
secretes pancreatic lipase, colipase, and cholesterol esterase
257
Micelles and Chylomicrons
Micelles are clusters of lipids soluble in the intestine, vital to digestion
Micelles diffuse to the brush border of the intestinal mucosal cells to be absorbed
-Chylomicrons leave the intestine via lacteals (lymphatic system) and re-enter via the thoracic duct
258
Lipid mobilization (hormone sensitive lipase and lipoprotein lipase)
a fall in insulin levels activates hormone sensitive lipase
hydrolyzes triacylglycerols, yielding fatty acids and glycerol
Epinephrine and cortisol can also activate HSL is effective within adipose cells, but lipoprotein lipase (LPL) is necessary for the metabolism of chylomicrons and very-low-density lipoproteins
can release free fatty acids from triacylglycerols
in these lipoproteins released glycerol from fat may be transported to the liver for glycolysis or gluconeogenesis
259
Lipoproteins, lipoproteins sorting
free fatty acids are transported through the blood with albumin, but triacylglycerol and cholesterol are transported in the blood as lipoproteins; aggregates of apolipoproteins and lipids
Sorted by density least to highest
-density increases in direct proportion to the percentage of protein in the particle
260
Lipoproteins- Chylomicrons
highly soluble in both lymphatic fluid and blood and function in the transport of dietary triacylglycerols, cholesterol, and cholesteryl esters to other tissues
261
Lipoproteins- VLDL (very-low-density lipoprotein)
metabolism similar to chylomicrons; however, VLDL is produced and assembled in liver cells
Main function is the transport of triacylglycerols to other tissues
262
Lipoproteins- IDL (intermediate-density lipoprotein)
once a triacylglycerol is removed from VLDL, the resulting particle is referred to as a VLDL remnant or IDL
exists as a transition particle between triacylglycerol transport (chylomicrons and VLDL) and cholesterol transport
263
Lipoproteins- LDL (low-density lipoprotein)
the majority of cholesterol in the blood is associated with LDL
264
Lipoproteins- HDL (high-density lipoprotein)
dense, protein-rich used to clean up excess cholesterol from blood vessels for excretion
265
Apolipoproteins
form the protein component of the lipoproteins, have diverse purposes
266
Sources of cholesterol, de novo synthesis, regulation
Most cells derive their cholesterol from LDL or HDL, but de novo synthesis of cholesterol does occur in the liver
driven by acetyl-CoA and ATP
Synthesis of mevalonic acid in the sER is the rate limiting step in cholesterol biosynthesis and is catalyzed by HMG CoA reductase
- increased levels of cholesterol can inhibit further synthesis
- insulin promotes cholesterol synthesis
267
LCAT (Lecithin-cholesterol acyltransferase)
an enzyme found in the bloodstream that is activated by HDL apoproteins
adds a fatty acid to cholesterol, which produces soluble cholesteryl esters such as those in HDL (these can be distributed to other lipoproteins)
-cholesteryl ester transfer protein (CETP) facilitates this transfer process
268
Essential fatty acids
alpha-linolenic acid and linoleic acid maintain cell membrane fluidity
269
fatty acid nomenclature
omega (ω) numbering system describes the position of the last double bond relative to the end of the chain and identifies the major precursor fatty acid
270
lipid and carbohydrate synthesis description
lipid and carbohydrate synthesis are often called nontemplate synthesis because they do not rely directly on the coding of a nucleic acid
271
Fatty acid biosynthesis
occurs in the liver, products are transported to adipose tissue
Enzymes _acetyl-CoA carboxylase and fatty acid synthase_
stimulated by insulin
palmitic acid (palmitate) is the primary end product of fatty acid synthesis
272
Acetyl-CoA shuttling
Citrate, a product of acetyl-CoA through the ETC, can diffuse across the mitochondrial membrane
-in the cytosol, citrate lyase splits citrate back into acetyl-CoA and oxaloacetate
273
Acetyl-CoA carboxylase
activates acetyl-CoA in the cytoplasm for incorporation into fatty acids
**rate-limiting enzyme of fatty acid biosynthesis**
requires biotin and ATP to function, and adds CO2 to acetyl-CoA to form malonyl-CoA
activated by insulin and citrate
274
Fatty acid synthase
palmitate synthase, palmitate is the only fatty acid humans can synthesize de novo
large complex found in the cytosol that is rapidly induced in the liver following a meal high in carbohydrates due to elevated insulin levels
requires pantothenic acid (vit B5) and NADPH and eight acetyl-CoA groups
275
Triacylglycerol (triglyceride) synthesis
addition of three fatty acids to glycerol primarily in the liver and somewhat in adipose tissue
276
beta oxidation location and regulation
B-oxidation occurs in the mitochondria and somewhat in peroxisomes
insulin indirectly inhibits while glucagon stimulates
277
beta oxidation- activation
when fatty acids are metabolized, they first become activated by attachment to CoA, which is catalyzed by fatty-acyl-CoA synthetase
278
beta oxidation- fatty acid entry into mitochondria
long-chain fatty acids require transport via a carnitine shuttle
## Footnote
**Carnitine acyltransferase I is the rate-limiting enzyme of fatty acid oxidation**
279
beta oxidation- in mitochondria (four steps)
beta-oxidation is the reverse of fatty acid synthesis
1 Oxidation of the fatty acid to form a double bond
2 Hydration of the double bond to form a hydroxyl group
3 Oxidation of the hydroxyl group to form a carbonyl (beta-ketoacid)
4 Splitting of the beta-ketoacid into a shorter acyl-CoA and acetyl-CoA
280
beta oxidation products
Each four-step cycle releases one acetyl-CoA and reduces NAD+ and FAD (producing NADH and FADH2) these go to ETC
acetyl-CoA enters the citric acid cycle in muscle and adipose tissue, in the liver it stimulates gluconeogenesis; much is used to synthesize ketone bodies
281
odd-numbered chain fatty acids beta oxidation differences
Odd-numbered chain fatty acids undergo the same process but with propionyl-CoA as the five carbon remaining fragment
-converted to glucose, exception to the rule that fatty acid is not converted to glucose
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Ketone bodies
acetoacetate and 3-hydroxybutyrate
283
ketogenesis
- occurs in the mitochondria of livers cells when excess acetyl-CoA accumulates in the fasting state
- HMG-CoA is formed and broken down into acetoacetate which can then be reduced to 3-hydroxybutyrate with acetone side product
284
ketolysis
3-hydroxybutyrate is oxidized to acetoacetate
-the liver lacks this enzyme
Ketolysis in the brain
-During a prolonged fast the brain derives up to two-thirds of its energy from ketone bodies
285
protein catabolism
protein is rarely used as an energy source but proteins must be digested and absorbed to provide a reservoir of amino acids
stomach- pepsin, trypsin, chymotrypsin, carboxypeptidases A and B
-zymogens
small intestine- dipeptidase and aminopeptidase
primarily muscle and liver amino acids released from proteins usually lose their amino group with the remaining carbon skeleton used for energy
-urea cycle removes the excess energy
286
relationship between enthalpy and heat exchange
At constant pressure and volume, enthalpy (deltaH) and thermodynamic heat exchange (Q) are equal ΔG = ΔH - TΔS
287
physiological conditions modified standard state
H+ = 10^-7 and the pH is 7
288
deltaG relation to Q equation
ΔG = ΔG\* + RTlnQ
289
flavoproteins
contain a modified B2 or riboflavin nucleic acid derivatives, present in the mitochondria and chloroplasts as electron carriers
involved in the modification of other B vitamins to active forms
function as coenzymes for enzymes in the oxidation of fatty acids
290
postprandial (absorptive) state
anabolic not catabolic
high insulin
glycogen synthesis in liver and muscle
excess glucose to fatty acids and triacylglycerols in liver
triacylglycerol synthesis in adipose tissue
nervous tissue and rbcs are insensitive to insulin, nervous tissue derives energy from oxidizing glucose to CO2
291
postabsorptive (fasting) state
glucagon, cortisol, epinephrine, norepinephrine, and growth hormone
opposite of absorptive state
292
prolonged fasting (starvation)
glucagon and epinephrine very elevated
gluconeogenic activity continues
gluconeogenesis is the predominant source of glucose
lipolysis is rapid, resulting in excess acetyl-CoA used in the synthesis of ketone bodies
-shift from glucose to ketones
--rbcs and cells w/o mitochondria still dependent on glucose
293
metabolic effects of insulin; tissue not affected
peptide hormone secreted by beta-islet cells of pancreas, key player in uptake and storage of glucose
increases rate of glucokinase and glycogen synthase in liver while decreases the activity of glycogen phosphorylase and glucose-6-phosphatase
increases glucose and triacylglycerol uptake by fat cells, lipoprotein lipase activity (clears VLDL and chylomicrons from the blood), triacylglycerol synthesis above 100mg/dL or 5.6
insulin secretion is proportional to plasma glucose
tissue not affected: nervous, kidney tubules, intestinal mucosa, rbcs, beta-cells of pancreas
294
metabolic effects of glucagon
peptide hormone secreted by the alpha-cells of the pancreatic islets of langerhans
increased liver glycogenolysis, activates glycogen phosphorylase and inactivates glycogen synthase
promotes the conversion of pyruvate to PEP by pyruvate carboxylase and phosphoenolpyruvate
increased ketogenesis decrease lipogenesis
increased lipolysis in the liver, activates hormone-sensitive lipase basic amino acids promote secretion secreted in response to protein rich meal
295
Glucocorticoids effect on metabolism
from the adrenal cortex, responsible for stress response, especially cortisol
promote mobility of energy
-elevates blood glucose and inhibits glucose uptake
296
metabolic effect of catecholamines
secreted by the adrenal medulla, includes epinephrine and norepinephrine
increase the activity of liver and muscle glycogen phosphorylase, promoting glycogenolysis and increasing glucose output
act on adipose tissue to increase lipolysis
increase basal metabolic rate
297
metabolic effect of thyroid hormones
action is largely permissive, levels are kept more or less constant
increase basal metabolic rate, T4 takes longer but is longer lasting
primarily lipid and carb metabolism
epinephrine requires thyroid hormones to have a significant metabolic effect
298
Tissue-specific metabolism - connective tissue and epithelium
little metabolism
299
Tissue-specific metabolism - liver
maintain a constant level of blood glucose and synthesize ketones when excess fatty acids are being oxidized
replenishes glycogen with excess glucose, any glucose remaining is converted to acetyl-CoA and used for fatty acid synthesis
300
Tissue-specific metabolism - adipose tissue
insulin triggers fatty acid release from VLDL and chylomicrons and can suppress the release of fatty acids
decreased insulin activate hormone-sensitive lipase in fat cells, releasing fatty acids
301
Tissue-specific metabolism - skeletal muscle (resting and activated)
resting muscle
- body’s major consumer of fuel, insulin promotes glucose uptake
- excess glucose and AAs can be oxidized for energy
- in fasting state, fatty acids derived from free fatty acids are utilized
activated muscle
- short lived energy from creatine phosphate, which transfers a phosphate group to ADP to form ATP
- after 1-3 hours of continuous exercise, muscle glycogen stores become depleted and intensity rate declines
302
Tissue-specific metabolism - cardiac muscle
unlike other tissues of the body, cardiac myocytes prefer fatty acids as their major fuel, even in the well-fed state
303
Tissue-specific metabolism - brain
consumes much of the glucose fatty acids cannot cross the blood-brain barrier, between meals, the brain relies on blood glucose supplied by either hepatic glycogenolysis or gluconeogenesis
Only during prolonged fasting does the brain gain the capacity to use ketone bodies for energy
304
Respirometry quotient
Respirometry quotient (RQ) indicates what fuels the body is using
RQ = CO2 produced/O2 consumed
305
Calorimeters
Calorimeters can measured BMR based on heat exchange
-can also be estimated based on age, weight, height, and gender
306
regulation of body mass
Lipids stored in adipocytes are the primary factor in the gradual change of body mass over time
Caloric changes has a threshold level that differs between individuals
307
ghrelin, orexin, leptin
Ghrelin - in response to signals of meals, increases appetite
orexin - further increases appetite also sleep-wake cycle
leptin - decreases appetite by suppressing orexin production
308
BMI
BMI = mass/height^2
309
D-fructose structure
310
D-glucose structure
311
D-galactose structure
312
D-mannose structure
