Amino acids proteins and protein synthesis + Bioanalysis and enzyme kinsases Flashcards
1
Q
What are the 4 categories of biomolecules?
A
`1. Proteins
2. Carbohydrates
3. Lipids
4. Nucleic acids
2
Q
- Protein functions include…
A
structural support,
storage, transport,
cellular
communications
, movement and defense
against foreign substances.
3
Q
What are Enzymatic proteins?
A
Function: Selective acceleration of
chemical reactions
Example: Digestive enzymes catalyze the
hydrolysis of bonds in food molecules.
4
Q
What are defensive proteins?
A
Function: Protection against disease
Example: Antibodies inactivate and help
destroy viruses and bacteria.
5
Q
What are storage proteins?
A
Function: Storage of amino acids
Examples: Casein, the protein of milk, is
the major source of amino acids for baby
mammals. Plants have storage proteins in
their seeds. Ovalbumin is the protein of
egg white, used as an amino acid source
for the developing embryo.at
6
Q
What are transport proteins?
A
Function: Transport of substances
Examples: Hemoglobin, the iron-containing
protein of vertebrate blood, transports
oxygen from the lungs to other parts of the
body. Other proteins transport molecules
across membranes, as shown here.
7
Q
What are hormonal proteins?
A
Function: Coordination of an organism’s
activities
Function: Response of cell to chemical
stimuli
Function: Movement Function: Support
Example: Insulin, a hormone secreted by
the pancreas, causes other tissues to take
up glucose, thus regulating blood sugar
8
Q
What are receptor proteins?
A
Function: Response of cell to chemical
stimuli
Example: Receptors built into the
membrane of a nerve cell detect signaling
molecules released by other nerve cells.
9
Q
What are contractile and motor proteins?
A
Function: Movement
Examples: Motor proteins are responsible
for the undulations of cilia and flagella.
Actin and myosin proteins are responsible
for the contraction of muscles.
10
Q
What are structural proteins?
A
Function: Support
Examples: Keratin is the protein of hair,
horns, feathers, and other skin appendages.
Insects and spiders use silk fibers to make
their cocoons and webs, respectively.
Collagen and elastin proteins provide a
fibrous framework in animal connective
tissues
11
Q
What happens at pH 7 for amino acids?
A
At pH 7 – the carboxyl group of an aa is in its conjugate
base form. Amino group in conjugate acid form.
12
Q
Amino acid diagram?
A
CHECK
13
Q
What’s the monomer and polymer?
A
Amino acids are the monomers
20 different types of amino acids each with their own R group
14
Q
What are Bioactive Amino Acids?
A
Several amino acids have important biological roles in addition to been essential to protein construction:
- Tryptophan is a precursor of the serotonin neurotransmitter.
- Tyrosine is a precursor of the dopamine neurotransmitter.
- Arginine is a precursor of nitric oxide.
- Aspartate, glutamine, glycine are precursors of nucleotides
15
Q
How do peptide bonds form?
(also look at the diagram)
A
Binding occurs between the amino group of one amino acid and the carboxyl group from another amino acid.
= Condensation reaction or dehydration synthesis
16
Q
What are amino acid residues?
A
Amino acid residues with free amino groups is called the N-terminal
and is written to the left
The free carboxyl group is on the C-terminal residue and appears to the right.
17
Q
Types of Peptides
A
Dipeptide = 2 aa
Tripeptide = 3 aa and so on.
Oligopeptides = <25 aa residues
Polypeptides = >25 aa residues
Proteins are oligomers containing 50 or more aa residues
18
Q
Proteins: history
A
Frederick Sanger –first to sequence a polypeptide (insulin) (~1943)
The sequence of amino acids in a protein offer
insights into its 3-D structure and its function,
cellular location and evolution.
19
Q
Protein structure and function
A
▪ The specific activities of proteins result
from their intricate three-dimensional
architecture
▪ A functional protein consists of one or
more polypeptides precisely twisted,
folded, and coiled into a unique shape
20
Q
Primary structure
A
Primary structure, the
sequence of amino acids
in a protein, is like the
order of letters in a long word.
Primary structure is
determined by inherited
genetic information.
21
Q
Secondary structure
A
The coils and folds of secondary structure result from
hydrogen bonds between repeating constituents of
the polypeptide backbone.
Typical secondary structures are:
1) a coil called an helix.
2) a folded structure called a pleated sheet.
22
Q
Tertiary structure
A
Tertiary structure is determined by interactions between
R groups, rather than interactions between backbone
constituents.
Hydrogen bonds, ionic bonds, hydrophobic interactions, van der Waals, covalent bonds (disulphide bridges)
23
Q
Quaternary structure
A
Quaternary structure results when two or more
polypeptide chains form one macromolecule.
24
Q
What is collagen and haemoglobin?
A
- Collagen is a fibrous protein
consisting of three polypeptides
coiled like a rope. - Haemoglobin is a globular protein
consisting of four polypeptides:
two alpha and two beta chains.
25
A change in primary structure…
▪ A slight change in primary structure can affect
protein structure and ability to function
▪ Sickle-cell disease, an inherited blood disorder,
results from a single amino acid substitution in the
protein hemoglobin
26
What determines protein structure?
▪ In addition to primary structure, physical and
chemical conditions can affect structure.
▪ Environmental factors: pH, salt concentration,
temperature can cause proteins to unravel
(denaturation).
▪ A denatured protein is biologically inactive.
27
How do we determine protein structure?
▪ X-ray crystallography.
▪ Nuclear magnetic resonance (NMR) spectroscopy,
which does not require protein crystallization.
▪ Bioinformatics is another approach to prediction of
protein structure from amino acid sequences.
28
Two hypotheses prevailed during the 1950s:
The peptide or multi-enzyme theory
The template theory
The peptide or multi-enzyme theory: proteins were assembled by
stepwise coupling of small peptides (of the amino acids), into polypeptide chains, a process guided by enzymes.
The template theory, which held that proteins were synthesized on
templates, one for each protein. The templates were genes.
29
The Roles of Nucleic Acids
▪ There are two types of nucleic acids
▪ Deoxyribonucleic acid (DNA)
▪ Ribonucleic acid (RNA)
▪ DNA provides directions for its own replication (Dr
Zubko).
▪ DNA (genes) directs synthesis of messenger RNA
(mRNA) which controls protein synthesis.
▪ This process is called gene expression:
transcription and translation
30
Basic Principles of Transcription and
Translation
▪ RNA is the bridge between genes and the proteins
for which they code.
▪ Transcription is the synthesis of RNA using
information in DNA.
▪ Transcription produces messenger RNA (mRNA).
▪ Translation is the synthesis of a polypeptide, using
information in the mRNA.
▪ Ribosomes are the sites of translation.
▪ A primary transcript is the initial RNA transcript
from any gene prior to processing.
31
Synthesis of an RNA Transcript
* The three stages of transcription
1) Initiation
2) Elongation
3) Termination
32
1) Molecular Components of Initiation
* Promoter: DNA sequence where RNA
polymerase attaches. TATA box in
Eukaryotes.
* Transcription factors: mediate binding of
RNA polymerase II.
+
* RNA polymerase II: pries the DNA strands
apart and links together the RNAnucleotides.
* Transcription initiation complex (RNA
polymerase II + transcription factors).
33
2) Elongation of the RNA Strand
▪ RNA polymerase unwinds the DNA double helix, 10
to 20 bases at a time. Links RNA nucleotides.
▪ mRNA is complementary to the DNA template
strand.
▪ Nucleotides are added to the 3′ end of the growing
RNA molecule
▪ Transcription rate of 40 nucleotides / s in Eukaryotes.
▪ RNA synthesis follows the same base-pairing rules as
DNA, except that uracil substitutes for thymine
34
3) Termination of Transcription
▪ Bacteria: polymerase stops transcription at the end
of the terminator and the mRNA can be translated
without further modification.
▪ Eukaryotes: RNA polymerase II transcribes the
polyadenylation signal sequence; the RNA transcript
is released 10–35 nucleotides after this poly-A
sequence / tail.
35
RNA processing (Eukaryotes)
1) Both ends of the primary transcript are altered.
2) Interior sections of the molecule can be excised and the remaining parts spliced together
3) Enzymes in the eukaryotic nucleus modify premRNA.
ALL OCCUR IN THE NUCLEUS OF THE CELL
36
1) Alteration of mRNA ends
▪ 5′ end receives a modified nucleotide 5′ (prime) cap.
▪ 3′ end has a poly-A tail.
37
Why does alteration of mRNA ends?
- Facilitate the export of mRNA to the cytoplasm.
- Protect mRNA from hydrolytic enzymes.
- Assist ribosomal attachment to the 5′ end.
38
2) RNA Splicing
▪ RNA splicing removes introns and joins exons,
creating an mRNA molecule with a continuous
coding sequence.
39
Introns – what is the point?
▪ Some contain sequences that may regulate gene
expression.
▪ Some genes encode more than one kind of
polypeptide, depending on which segments are
treated as exons during splicing.
▪ This is called alternative RNA splicing
Which means: Number of different proteins an organism can
produce is much greater than its number of genes
40
The Genetic Codec
▪ There are 20 amino acids, but there are only four
nucleotide bases in DNA (A, T, C, G).
41
What are codons
a sequence of three nucleotides which together form a unit of genetic code in a DNA or RNA molecule.
42
The Triplet Code cracking the code?
▪ 64 codons (61 code for amino acids; 3 “stop”
signals to end translation).
▪ More than one codon may specify a particular amino
acid but no codon specifies more than one amino
acid.
▪ Codons must be read in the correct reading frame
(correct groupings) in order for the specified
polypeptide to be produced.
43
Transfer RNA (tRNA)
▪ These adaptor molecules are called transfer RNA
(tRNA).
▪ tRNAs transfer amino acids to the growing
polypeptide in a ribosome.
▪ RNA molecules, 80 nucleotides long.
▪ Flattened into one plane to reveal its base pairing
44
Accurate translation requires two steps:
(1) a correct match between a tRNA and an amino
acid, performed by the enzyme aminoacyl-tRNA
synthetase.
(2) a correct match between the tRNA anticodon
and an mRNA codon.
45
The Structure and Function of Ribosomes
▪ Ribosomes facilitate specific coupling of tRNA
anticodons with mRNA codons in protein synthesis.
▪ The two ribosomal subunits (large and small) are
made of proteins and ribosomal RNA (rRNA).
▪ Bacterial and eukaryotic ribosomes differ.
▪ 2x ribosomal subunits.
▪ 3x binding sites (A, P, E)
46
Building a Polypeptide
What are the 3 stages of translation?
All 3 stages require what?
What's required for the steps?
▪ The three stages of translation:
1) Initiation
2) Elongation
3) Termination
▪ All three stages require protein “factors” that aid in
the translation process
▪ Energy is required for some steps
47
Molecular Components of Translation
▪ mRNA
▪ tRNAs (with amino acid added)
▪ Ribosome (large and small subunits): made of
protein and rRNA molecules.
▪ Translation factors
▪ Start codon (AUG)
▪ Stop codons
48
1) Initiation
2)Elongation
3) Termination
1) Codon recognition = I
2) Peptide bond formation = E
3) Translocation = E
4) Ribosome reaches a stop codon on mRNA.
5) Release factor promotes hydrolysis.
6) Ribosomal subunits and other components dissociate.
49
Why is a Polyribosome
(or polysome). helpful
Produce polypeptides / proteins quickly
50
Function of the nuclear envelope in transcription and translation?
▪ Eukaryotes have the nuclear envelope which
separate transcription (nucleus) and translation
(cytoplasm / ER).
▪ RNA undergoes processing before leaving the
nucleus.
51
Completing and targeting the functional protein
Often translation is not sufficient to make a functional
protein:
- Polypeptide chains are modified after translation
(post-translational modification), maybe required
for correct folding (tertiary structure etc).
- Protein targeting to specific sites in the cell (use of
signal peptide) such as the ER.
52
What is analytical chemistry?
a branch of chemistry that deals with
the identification of compounds and mixtures (qualitative analysis)
or the determination of the proportions of the constituents
(quantitative analysis): techniques commonly used are titration,
precipitation, spectroscopy, chromatography, etc
53
What is biochemical analysis?
characterisation of biological components within
a sample using appropriate laboratory techniques
54
What is Qualitative Analysis
indicate whether a particular substance (analyte)
is present above a threshold level
55
What is Quantitative Analysis:
determines the amount of a particular analyte
present in the sample (e.g. the concentration of a drug in blood serum).
56
Definition of
Accuracy
Precision
Accuracy: Closeness of a measured or derived data value to its true value
Precision: Closeness of values with repeated measurements
57
What is
sensitivity
Specificity
Sensitivity:
* ability to detect small amounts of analyte in a sample
* OR the percentage of patients with the disease that will be correctly
identified as disease positive
Specificity:
* ability to detect only the analyte of interest in a sample
* OR the percentage of patients without the disease that receive a
negative result
58
Typical analytical scheme?
Sample
Sub-sample
Extraction
Separation
Detection
Result
Interpretation
59
What is chromatography and how's it discovered?
Technique that allows the resolution of a mixture of
compounds as a consequence of the different rates at
which they move through a stationary phase, under the
influence of a mobile phase
Discovered by Mikhail Tsvet = separating plant pigments by
extracting them from leaves with ether
and alcohol and percolating the solution
through a column of calcium carbonate
60
Whats Planar chromatography?
Planar Chromatography:
The stationary phase is supported on a flat plate or in the fibres of a paper.
The mobile phase moves through the stationary phase by capillary action
or by gravity.
* Paper chromatography
* Thin layer chromatography (TLC)
61
Whats column chromatography?
Column Chromatography:
The stationary phase is held in a tube through which the mobile phase is forced either by pressure or by gravity.
* Simple column chromatography
* High pressure liquid chromatography (HPLC)
* Gas chromatography (GC)
62
Thin Layer Chromatography
TLC plate (stationary phase): a sheet of
glass, metal, or plastic coated with a thin
layer of a solid adsorbent (usually silica or
alumina)
Samples are “spotted” at the base of the
sheet and dried.
Sheet is placed in the tank containing a
shallow layer of solvent (mobile phase)
63
Retention factor formula?
distance travelled by substance /
distance travelled by solvent
64
Whats gel filtration ( column chromatography)
Separates proteins, peptides and
oligonucleotides on the basis of size
The stationary phase (gel) consists of
beads (e.g. Sephadex)
The mobile phase (buffer) is used to
elute the analytes from the gel matrix
based on their size
65
Gel filtration formula?
Vt = Vs + VI + Vo
Vo: volume required to elute
molecules bigger than the pore
size of the column gel
VI= volume of solvent in the pores
Ve: elution volume of a
particular solute
66
Partition coefficient?
Partition coefficient:
Kav = (Ve - Vo) / (Vt -Vo)
67
Spectroscopy
Measures the absorption and emission of electromagnetic radiation by
atoms and molecules in solution
Isaac Newton (1666):
uses a glass prism to split sunlight into
a monochrome spectrum
68
Spectroscopy: Beer-Lambert law
Describes the absorbance of monochromatic light as it passes through a solution
A = εcl
Absorbance is linearly related to the concentration of the analyte
69
Whats metabolism?
* Metabolism is the sum total of the chemical processes that occur in living
organisms, resulting in growth, production of energy, elimination of waste
material, etc
70
Whats a metabolic pathway?
* A metabolic pathway begins with a specific molecule and ends with a product. Each step is catalysed by a specific enzyme
71
What is a chemical reaction?
Chemical reactions involve the transfer of energy.
Every chemical reaction between molecules involves bonds breaking and bonds forming.
Exergonic reactions result in a net release of free energy but require Activation energy (EA) to initiate the reaction.
72
Do exergonic reactions require energy?
Exergonic reactions don’t require energy input beyond the activation energy. This energy
can often be obtained environmentally from heat so the reaction will occur
spontaneously.
73
Endergonic reactions?
Endergonic reaction require relatively large amounts of energy to occur, so does not occur spontaneously.
74
How do exergonic and endergonic reactions relate to biology
* Catabolic pathways (mostly exergonic) release
energy by breaking down complex molecules into
simpler compounds
Cellular respiration, the breakdown of glucose
in the presence of oxygen, is an example of a pathway of
catabolism
* Anabolic pathways (endergonic) consume energy
to build complex molecules from simpler ones
For example, the synthesis of protein from amino acids
is an anabolic pathway
75
Introduction of enzymes in chemical reactions
The introduction of an enzyme reduces the activation energy required and speeds up
the reaction.
76
Enzyme characteristics
The enzymes are:
▪ Organic molecules (proteins or RNAs), which speed
up (catalyse) chemical reactions by up to 1012-fold
▪ Highly specific – work only on a particular substrate
▪ Unaffected by the reaction they catalyse
▪ Can catalyse the same chemical reaction in the opposite direction
▪ The enzyme activity can be regulated
77
The active site can lower the activation
energy needed by a reaction by:
* orienting substrates correctly
*straining substrate bonds
* providing a favorable microenvironment
* covalently bonding to the substrate
78
Enzyme cofactors what are they
* Cofactors are nonprotein
enzyme helpers
* Cofactors may be inorganic
(such as a metal in ionic form)
or organic
* An organic cofactor is called a
coenzyme
* Coenzymes include vitamins
79
Some Enzymes Require Cofactors
▪ Other enzymes are composed of apoenzymes (a protein portion)
and one or more nonprotein cofactors
▪ The combination of both apoenzyme and its cofactors is a
holoenzyme
80
Co factor enzymes
▪ Inorganic cofactors (metal ions) include Fe, Mg, Zn, or Cu ions
* Co-substrates: associate transiently with an enzyme to catalyse a
reaction: include vitamins (e.g. NAD+, NADP+, and FAD)
* Prosthetic groups (non-polypeptides): associated permanently
with enzymes with a covalent bond
81
The importance of coenzymes
Deficiencies in enzyme cofactors can result in a number
of different medical conditions.
82
What is “rate of a chemical reaction”?
The concentration of a product that is
formed in a unit of time or the
concentration of a substrate that is
consumed in a unit of time.
83
Initial rate (velocity) of a reaction (V0):
a measure of the activity
(reaction rate) of an enzyme
84
Why does V0 slow down?
▪ Vo slows as enzyme loses
activity or substrate [S] levels
decrease
85
When the substrate is in excess
→ little change in V0
(the enzyme is
occupied nearly 100%)
maximal velocity is reached
86
What is Michaelis constant (Km)
▪ Michaelis constant (Km): the
substrate concentration at which
Vo is half maximum
87
Enzyme inhibition and regulation
Enzyme inhibitors affect the
rate at which an enzyme
processes its substrate by
directly interacting with the
enzyme.
Enzyme regulation involves
both inhibitors and activators.
88
Michaelis-Menten equation:
rate equation for a one substrate enzyme catalysed reaction
89
What are irreversible inhibitors?
▪ Irreversible inhibitors : bind covalently to the active site of an
enzyme (e.g. Aspirin irreversibly inhibits COX enzymes)
90
What are Reversible inhibitors?
▪ Reversible inhibitors: form weak bonds with the enzyme →
rapid dissociation of the enzyme: inhibitor complex
91
What is a competitive inhibitors?
* Competitive inhibitors:
- resemble the substrate
- bind to the active site of an enzyme
92
What are non competitive inhibitors?
* Noncompetitive inhibitors:
- bind at a site distinct (allosteric site)
from the active site of an enzyme
- change the conformation of the enzyme
and its active site
93
Allosteric regulation
Allosteric regulation may either inhibit or stimulate an enzyme’s activity
* Most allosterically regulated
enzymes are made from
polypeptide subunits, each with its
own active site
* The enzyme complex has active and
inactive forms
* The binding of an activator
stabilizes the active form of the
enzyme
* The binding of an inhibitor stabilizes
the inactive form of the enzyme
94
What is cooperativity
* Cooperativity is a form of allosteric
regulation that can amplify enzyme activity
* One substrate molecule primes an enzyme
to act on additional substrate molecules
more readily
* Cooperativity is allosteric because binding
by a substrate to one active site affects
catalysis in a different active site
95
What is feedback inhibition?
* In feedback inhibition, the
end product of a metabolic
pathway shuts down the
pathway
* Feedback inhibition prevents
a cell from wasting chemical
resources by synthesizing
more product than is needed
96
Inhibitor effect on v max
▪ Competitive inhibitors: Km is increased but the Vmax is unaltered
▪ Non-competitive inhibitors : Vmax is reduced – but the Km is
unaltered
