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BCHM 270 STUDY GUIDE WITH
COMPLETE SOLUTION
Nucleotides (definition) - ANSWER basic structural unit for DNA and RNA (called a nucleoside if they do not contain phosphate groups)
Nucleotides (structure) - ANSWER 1) Nitrogenous base
- Purines: two rings - adenine, guanine
- Pyrimidines: one ring - cytosine, uracil, thymine
- DNA uses adenine, guanine, cytosine, and thymine
- RNA uses adenine, guanine, cytosine, and uracil
- 5-carbon sugar
- Deoxyribose: no hydroxyl group on the second carbon
- Ribose: hydroxyl group on the second carbon
- DNA uses deoxyribose
- RNA uses ribose
- Phosphate
- Up to three phosphate groups can be present in mononucleotides
- When bound in DNA/RNA, one phosphate group forms the backbone of the structure
DNA (structure) - ANSWER The DNA backbone is formed by phosphodiester
bonds (dehydration reactions) between the phosphate group of one nucleotide and the hydroxyl group on the 3' carbon of another nucleotide
Two DNA strands are held together by hydrogen bonds formed between complementary base pairs
- AT forms 2 hydrogen bonds
- CG forms 3 hydrogen bonds, so is stronger
DNA mutations - ANSWER Polymorphisms: variations in characteristics (eg. eye colour, hair colour) due to alleles (variant genes)
Point mutations: single nucleotide changes
- Silent mutation: amino acid is not changed
- Missense mutation: amino acid is changed
- Nonsense mutation: premature stop codon results in a smaller protein
- Frameshift mutation: an insertion or deletion shifts the enter amino acid sequence
Functional mutations: mutations are classified based on how they affect the function of the protein
- Gain of function mutation
- Loss of function mutation
- Dominant negative function mutation: dysfunctional proteins also reduce the function of the nun-mutated allele's protein
RNA (structure) - ANSWER - Single stranded
Glycine - ANSWER Gly, G
smallest side chain (hydrogen), which adds flexibility to the peptide chain
Alanine - ANSWER Ala, A
side chain contains C and H
Valine - ANSWER Val, V
side chain contains C and H
Phenylalanine - ANSWER Phe, F
contain a benzene ring (aromatic)
Leucine - ANSWER Leu, L
side chain contains C and H
Isoleucine - ANSWER Ile, I
side chain contains C and H
Tryptophan - ANSWER Trp, W
contain a benzene ring (aromatic)
Methionine - ANSWER Met, M
contains a sulfur along with carbon and hydrogen on its side chain
Proline - ANSWER Pro, P
R group is bound to amino group, creating a rigid kink in amino acid chain
Serine - ANSWER Ser, S
have a hydroxyl group capable of forming hydrogen bonds
Threonine - ANSWER Thr, T
have a hydroxyl group capable of forming hydrogen bonds
Tyrosine - ANSWER Tyr, Y
has a hydroxyl group capable of forming hydrogen bonds, aromatic, and OH can lose a proton at alkaline pH
Asparagine - ANSWER Asn, N
has a carbonyl and amine that can both form H bonds and dipoles
Glutamine - ANSWER Gln, Q
has a carbonyl and amine that can both form H bonds and dipoles
Cysteine - ANSWER Cys, C
has a thiol group (-SH), can form disulfide bonds between each other, can lose a proton at alkaline pH
Aspartic acid - ANSWER Asp, D
Contains COO-
Glutamic acid - ANSWER Glu, E
Contains COO-
Histidine - ANSWER His, H
Contains aminobenzene
Lysine - ANSWER Lys, K
Contains amino group
bonds with a fully extended backbone and side chains above and below the strands - strands can be parallel or antiparallel to each other
Motifs: combinations of alpha helices and beta sheets forming supersecondary structures
Protein structure (tertiary) - ANSWER Folding of domains together to form a 3D structure stabilized by disulfide bonds, hydrogen bonds between side chains with FON groups, and ionic interactions between charged side chains
Protein structure (quaternary) - ANSWER Binding of 2+ identical/different protein subunits together, stabilized by noncovalent interactions and sometimes disulfide bonds
Post-translational modifications - ANSWER - Disulfide bonds: cysteine thiol group forms a covalent disulfide bond with other cysteines (thiol must be oxidized on both cysteines), helps stabilize protein
- Glycosylation: carbohydrate is added, either to amie group in asparagine (N-linked) or to the hydroxyl group in serine/threonine (O-linked)
- Phosphorylation: phosphate added to the hydroxyl group in serine, threonine and tyrosine to change the function or activate a protein - added by kinases and removed by phosphatases
Protein shapes - ANSWER - Globular proteins: roughly spherical, with hydrophobic cores and hydrophilic exteriors; some contain quaternary structure - serve as molecular transporters, storage proteins, enzymes, etc
- Fibrous proteins: long chains of multimers running parallel to each other and often twisted together; often stabilized by disulfide bridges - play a structural role in the body
Hemeproteins - ANSWER Globular family of proteins that contain heme as a tightly bound prosthetic group (not a protein)
- has many functions depending on the protein it is bound to: it can be an electron carrier, held remove hydrogen peroxide, and reversibly bind to oxygen
Myoglobin (Mb) - ANSWER Heme protein: oxygen carrier to heart and skeletal muscle
- Single peptide chain that is mostly an alpha helix, interior is nonpolar and exterior is polar
- Heme binds to a pocket lined with nonpolar amino acids - distal His binds to Fe of heme and distal His stabilizes binding of oxygen to Fe of heme
- Each Mb can reversibly bind to a single oxygen - the amount of oxygen bound to Mb in the blood is represented by percentage leading to a hyperbolic binding curve
- Mb always binds oxygen with the same affinity, so it is usually 90%
Restriction Endonucleases - ANSWER Enzymes naturally produced by bacteria which cleave double-stranded DNA at 4-6 bp palindromes to produce "restriction fragments" - scientists can use these enzymes for DNA cloning. Cuts are done to generate a 3' end with a hydroxy terminus and a 5' end phosphate on the other end. Cuts can be "sticky" (staggered) or "blunt" (ends will not hydrogen bond with each other)
Nomenclature of enzymes:
- First letter is the genus of the bacterium
- Next 2 letters are the species of the bacterium
- Number is the order in which the enzyme was discovered per organism
PCR - ANSWER Used to amplify a select DNA sequence in order to compare genes, detect sequences, or for forensic analysis. DNA is heated to 95 ° C to denature the strands, then cooled to 55-60 ° C so complementary primers can anneal, then increased to 65 ° C so Taq DNA polymerase can synthesize complementary strands.
Allele-specific PCR can determine single-nucleotide polymorphisms (SNP) by putting primers for wild/mutant alleles and then determining the length of promoted DNA using gel electrophoresis
Gel Electrophoresis - ANSWER Method used to separate molecules by size (done by separating by charge) - often done with restriction digest fragments and PCR products in order to visualize them. Samples are placed on agarose/polyacrylamide gels and then an electric field is applied. The larger the molecule, the less distance it will travel. The gel is stained with fluorescent ethidium bromide in order to visualize the distance of the molecules.
DNA Probes - ANSWER Single stranded DNA labelled with radioisotopes or fluorescent dye, complementary to target DNA. They are bound to the target DNA (hybridization) in order to identify target sequences.
They can also be designed to only bind to one version of an allele - allele specific oligonucleotides (ASO). This can be used with PCR to report on whether samples contain certain mutations or polymorphisms.
Southern Blotting - ANSWER Transfer of macromolecules onto a solid-phase membranous support such as nylon or nitrocellulose. This is done after gel electrophoresis and before hybridization in order to visualize results of these tests.
Southern blotting refers to DNA blotting.
From here, the process is identical to regular PCR
cDNA Microarray - ANSWER Normal and mutant genes are probed with different fluorescent compounds and visualized at once to determine gene sequences
Performed with mRNA reverse transcribed to cDNA.
SDS-PAGE - ANSWER Proteins are unfolded by an anionic detergent (SDS), dyed, and then run through electrophoresis to separate by size
Antibodies - ANSWER Antibodies are conjugated with enzymes or fluorescent tags in order to detect their antigen with high specificity. Used for protein purification, identification, quantification, or localization within a cell of interest.
Western Blotting - ANSWER Transfer of macromolecules onto a solid-phase membranous support such as nylon or nitrocellulose. This is done after gel electrophoresis and before hybridization in order to visualize results of these tests.
Western blotting refers to protein blotting. They use antigen-specific enzyme-labelled antibodies to confirm the presence and size of the protein on the gel.
ELISA - ANSWER Direct ELISA: wells are coated with an antigen of interested and then samples are tested for antibodies - used to check for immunity
Sandwich ELISA: wells are coated with the antibody, samples of unknown amount of antigen are added to the wells, secondary antibody is added connected to a colour substrate to visualize presence of the antigen (quantitative)
Acidity of amino acids - ANSWER - All free amino acids have two ionizable groups (can participate in acid/base reactions): weak acid (carboxyl group) and weak base (amino group)
- R-groups of acidic and basic amino acids can also act as weak acids/bases
For nonionizable R groups, both carboxylic group (pK1 = 2.3) and amino group in amino acid act as buffering groups (pK2 = 9.1) by becoming deprotonated
- When one group is protonated and the other is not, the molecule is in its zwitterionic form
- Isoelectric point (pI): pH where the net charge on a molecule is equal to 0
Active site models - ANSWER An active site is made up of a binding site and a catalytic site - there are 2 models in which this could occur:
- Lock and key model: each enzyme has a unique shape to fit its substrate - explains the specificity of binding but not enzyme flexibility, transition state stabilization, or how catalysis occurs
- Induced fit model: active site is constantly reshaped by interactions with substrate and enzyme, stabilizing the substrate into a transition state and allowing the reaction to move forward
Gibbs free energy - ANSWER combination of enthalpy, entropy, and temperature, and it is used to predict if a reaction will occur spontaneously or not:
G = H - TS
Endergonic reaction - ANSWER absorbs energy from surroundings into system (must be coupled with exergonic reactions in order to be spontaneous)
Exergonic reaction - ANSWER releases energy from the system to the surroundings (spontaneous)
Enzyme reaction energy - ANSWER Catalysts lower activation energy to increase reaction rate, but activation energy is not involved in Gibbs free energy so it does not influence whether a reaction is spontaneous or not
- Enzyme-substrate (ES) complex bonding causes a slight decrease in free energy
- ES complex undergoes catalysis, activation energy is lower than normal
- Formation of enzyme-product (EP) complex causes a slight decrease in free energy
- Product is released from enzyme, slightly increasing free energy
Enzyme assumptions - ANSWER 1. Values must be measured when the substrate concentration is much greater than the enzyme concentration
- Values must be measured when the reaction is well below maximum velocity
- Values must be measured as soon as possible after substrate/enzyme mixing to avoid product inhibition
- If reaction is measured multiple times, the same enzyme concentration must be used
Factors that impact enzyme activity - ANSWER - Temperature: enzyme activity increases with temperature until the point of denaturation; most enzymes are most active between 30 - 37 ° C
- Noncompetitive inhibitors: bind to bound and unbound enzyme at allosteric site (decrease Vmax)
- Mixed inhibitors: bind to bound and unbound enzyme at allosteric site (decrease Vmax, increase/decrease Km)
- Irreversible inhibitors: bind permanently
Michaelis-menten equation - ANSWER unregulated enzymes
Vo = (Vmax[S])/(Km + [S])
Lineweaver plot equation - ANSWER Reciprocal of michaelis-menten equation
- Km is on the x-intercept
- Vmax is on the y-intercept
1/Vo = Km/Vmax[S] + 1/Vmax
Hill equation - ANSWER allosteric enzymes
Vo = (Vmax[S]^n)/(K1/2^n + [S]^n)
ATP - ANSWER - Composed of an adenosine molecule, a ribose sugar, and a chain of three phosphates
- Standard free energy stored between the phosphate bonds is -7.3 kcal/mol
- Reactions couple with the ATP-ADP reaction in order to become overall favourable
- ATP can also be an allosteric factor
- GTP is identical except the adenosine is replaced with guanosine
Flavin adenine dinucleotide (FAD) - ANSWER Changed to high energy form by the addition of two H+ ions and two electrons, producing FADH
Nicotinamide adenine dinucleotide (NAD+) - ANSWER changed to high energy form by the addition of an H+ ion and two electrons, producing NADH
Nicotinamide adenine nucleotide phosphate (NADP+) - ANSWER - High energy form NADPH is an analog to NADH
- used in anabolic processes such as gluconeogenesis and lipid synthesis
- Extra phosphate does not affect chemical reaction activity, but is effective at changing the substrate binding of enzymes
TCA Cycle - ANSWER - Occurs in the mitochondria of all cells with
