Structure and function of proteins
- Secondary structure:
Hydrogen bond: between O of CO group and H of NH group - Tertiary structure
Ionic bond: between positively charged R group
Hydrogen group: between polar R group
Function of protein:
- Provide energy for our body, through food
General characteristics of enzyme function, regulation of enzymes
- General characteristics
Enzyme is the catalysis of biological systems, help speed up the reactions without itself undergoing any permanent chemical changes
a. Immense catalytic power: They are very active at small quantities. A small amount of enzymes can catalyze a large amount of substrates. The enzymes remain unchanged after the reaction
b. Highly specific: Enzymes are highly specific with their choice of substrates and catalyzed reaction. They only catalyzed 1 reactions or closed set of reactions. They are specific and only act on particular substrates that fit into their active site. This mechanism is the same with “Lock and key”: 1 lock can only be opened by 1specific key. Nearly every chemical reaction requires a specific enzyme
The reason for this characteristics is due to the precise interaction of the enzyme and substrate. This precision is the result of the complex 3D structure of enzyme.
c. Reversibility: Most of the enzyme-catalyzed reactions are reversible. The reversibility of reactions depends upon the requirements of the cells. In some cases, there are separate enzymes for forward and reverse reaction. Some reactions use the same enzyme for both.
d. Sensitive with heat, temperature and pH: The enzymes are thermolabile, means that they are easily to be destroyed by heat. Enzymes are inactive at very low temperature and be denatured at a really high temperture. Each enzyme has their own optimal temperature and pH to be functioned.
e. Can be inhibited by inhibitors: Sometimes it is better to slow down the ability of enzyme in catalyzing reactions.
f. Some can only active with the help of cofactors: Some enzymes have to attach to cofactors ( a non-protein molecule) to start functioning.
h. They speed up the biological reactions
i. They can be used again and again because after catalyzing a reaction, their chemical structure doesnt change - Regulation of enzymes
By regulating or controlling enzymes, the rate of reactions can be controlled. There are 5 mechanisms of regulating an enzyme.
a. Allosteric regulation: A regulator molecule (activator or inhibitor) binds to an enzyme in a site which is different from their active site. That site is called “Allosteric site”.
- Allosteric enzymes and protein can display “cooperativity” –> The binding on 1 site alter the other site binding affinity for binding.
- Enzyme activity in allosteric regulation is very sensitive to the change of substrate concentration.
b. Covalent modification:
- The activity of enzymes can be changed by covalent modifying structure. It caused the interconversion between the active and inactive site of an enzyme.
-An example is phosphorylation. The enzyme kinase catalyses the transfer of 1 phosphoryl group from ATP onto an enzyme, before attach that phosphoryl group into another molecule
c. Activation by limited proteolysis: Some enzymes are synthesized by inactive precursors (zymogen or proenzyme) that are subsequently activated by specific cleavage of peptide bonds. Once activated, they can eventually inactivated by the binding of inhibitors.
Example: Digestive enzyme
d. Enzyme concentration: regulates the amount of enzymes present. By regulating the transcription of specific genes, we can control how much enzyme is produced. This can in turn control the activity and functionality level of that enzyme.
e. Isoenzymes: They are enzymes that have different aa sequences with the enzyme but catalyses the same reaction. They usually exhibit different enzyme kinetics (Vmax, Km…) and are controlled by different regulatory molecules.
Kinetic parameters of enzyme function, the Michaelis-Menten model
Saturated —> All active sites are occupied. Adding more substrate makes no different to the reaction rate.
The graph explaination:
1. At the beginning, it is a straight line and a first order reaction. This implies that the rate of reaction is proportional to the substrate concentration.
2. Towards to end, [S] is very high —> starting to reach the highest point Vmax —> Zeroth order reaction. This means increasing the substrate concentration will not effect the grade.
Enzymes have varying tendencies to bind their substrates (affinities). An enzyme’s Km describes the substrate concentration at which half the enzyme’s active sites are occupied by substrate. A high Km means a lot of substrate must be present to half-saturate the enzyme, meaning the enzyme has low affinity for the substrate. On the other hand, a low Km means only a small amount of substrate is needed to half- saturate the enzyme, indicating a high affinity for substrate.
There is a relationship between KM, Vmax and inhibitors
1. Competitive inhibitors: impair reaction progress by binding to an enzyme, often at the active site, and preventing the real substrate from binding. At any given time, only the competitive inhibitor or the substrate can be bound to the enzyme (not both). That is, the inhibitor and substrate compete for the enzyme. Competitive inhibition acts by decreasing the number of enzyme molecules available to bind the substrate.
-> With a competitive inhibitor, the reaction can eventually reach its normal Vmax, but it takes a higher concentration of substrate to get it there. In other words, Vmax is unchanged, but the apparent Km is higher. In order to reach Vmax, more substrates are added to overcome the inhibitors
- Noncompetitive inhibitor: don’t prevent the substrate from binding to the enzyme. However, when the inhibitor is bound, the enzyme cannot catalyze its reaction to produce a product. Thus, noncompetitive inhibition acts by reducing the number of functional enzyme molecules that can carry out a reaction.
=>With a noncompetitive inhibitor, the reaction can never reach its normal Vmax, regardless of how much substrate we add. A subset of the enzyme molecules will always be “poisoned” by the inhibitor, so the effective concentration of enzyme (which determines Vmax) is reduced. However, the reaction reaches half of its new Vmax at the same substrate concentration, so Km is unchanged. The unchanged Km reflects that the inhibitor doesn’t affect binding of enzyme to substrate, just lowers the concentration of usable enzyme.
k2 or kcat, is the turnover number of the enzyme
Enzyme inhibition, types of inhibition
Competitive inhibition acts by decreasing the number of enzyme molecules available to bind the substrate
noncompetitive inhibition acts by reducing the number of functional enzyme molecules that can carry out a reaction.
Noncompetitive inhibition: Noncompetitive inhibitors change the shape of the active site —> Substrate can still bind to enzyme but the reaction is blocked
The structure and function of biological membranes
The two halves of the lipid bilayer are called leaflets
Basics of glycobiology
Glycobiology: The study of carbohydrates (structure, biosynthesis,…)
Carbohydrates: carbon-based molecules that are rich in hydroxyl group
1. Classifcation based on carbonyl group ( Carbonyl group composes of a carbon atom double-bonded to an oyxgen atom)
- Aldose: in its open form, contains aldehyde functional group => carbonyl group is attached to a carbon atom at the end of a carbon chain (R-CHO), R group can be H or any length carbon chain
Example: Glucose
- Ketose: in its open form, contains ketone functional group => carbonyl group is attached to a carbon atom within the carbon chain (RCOR’), R and R’ must be carbon chain
Example: Fructose
2. Classfication based on hydrolysable (How they undergo hydrolysis)
a. Monosaccharide: Simplest carbohydrate. Cannot be hydrolyzed to simpler compounds
- Trioses (3C): Glyceraldehyde => Takes part in glycolysis, cellular respiration
- Pentoses (5C): Ribose => Constituent of DNA, Deoxyribose ==> Consituent of RNA
- Hexoses (6C): Glucose => Takes part in many cell metabolism (Glycolysis, Glycogen metabolism…), Fructose ==> Fruit sugar, has a low impact on blood glucose levels
b. Disaccharide:
- is formed by 2 monosaccharides linked together by glycosidic bond, undergo condensation
Only sugars with the cyclic forms have an anomeric carbon and are capable of forming a glycosidic link. An anomeric carbon can be identified as the carbonyl carbon in the open-chain form of sugar.
- The bond between a OH group on 1 sugar and the anomeric C on other sugar
- alpha - glycosidic bond: OH group of the 1st monosaccharide in the 1st anomeric carbon is below the glucose ring, pointed down
- beta - glycosidic bond: OH group of the 1st monosaccharide in the 1st anomeric carbon is above the glucose ring, pointed up => The bond formed is a zig-zag bond
- Example: Glucose + Glucose –> Maltose (alpha)
Glucose + Galactose –> Lactose (Beta)
Glucose + Fructose –> Sucrose (alpha)
c. Polysaccharide: contains hundred or thousands of monosaccharide
Vital roles in energy storage and in maintaining the structural integrity of an organism
- Storage polysaccharide:
- Starch: store of glucose in plants (D-glucose)
- Glycogen: store of glucose in animals, human (alpha-D-glucose)
- Structural polysaccharide:
- Chitin: in cell wal of fungi
- Cellulose: in cell wall of plant, very stable (beta-D-glucose)
- Special type: Homopolymer => The monosaccharides in a polysaccharide are all the same
3. Classification based in chemical property
- Hemiacetal: Have OH group and OR group attached to the same Carbon
- Acetal: two O-R group attached to the same carbon
a. Reducing sugar (Lactose, Maltose)
- Has a free hemiacetal that can open up to form a free aldehyde group that can be oxidized under specific conditions => The aldehyde group is oxidized to a carboxylic acid. It can reduce another compound => Reducing agent
- The anomeric carbon is a hemiacetal. The carbon bonds to the ring oxygen and a OH group
b. Non-reducing sugar (Sucrose)
- Has no free hemiacetal to transform into a free aldehyde and so will not be oxidized under specific conditions => Cannot reduce another compound
- The anomeric carbon is acetal. The carbon doesn’t bond to any OH group, it bonds to 2 oxygen-containing groups
- Stereoisomerism
- Carbohydrates have one or more chiral centers- carbons with 4 different groups attached, giving rise to the possibility of stereoisomers. Generally, a compound containing “n” chiral centers can maximally have 2“n” stereoisomers.
- Stereoisomer: same molecular formula, same structure but different in spatial arrangement (orientations of atoms in space)
Example: cis and trans
- Enatiomers: stereoisomers whose structures are non-superimposible mirror images of each other (they cannot mentally merged into one object as they are brought together) => Identical in physical properties
- Diastereomers: stereoisomers that are not mirror images of each other => Different in physical properties
Glycolysis and its regulation
Citric acid cycle and its regulation
A series of chemical reactions to release stored energy through the oxidation of acetyl-CoA derived from carbohydrates, fats, and proteins. In addition, the cycle provides precursors of certain amino acids, as well as the reducing agent NADH, that are used in numerous other reactions.
Terminal oxidation and oxidative phosphorylation
Cytochrome: heme complex, Fe core
Matrix has low H+, Intermembrane has high H+
Glycolysis: Glucose –> Pyruvate: 2 ATP, 2 NADH, 2H+
Oxidation of pyruvate: Pyruvate –> Acetyl-CoA –> 2 CO2, 2NADH, 2H+
Krebs cycle: 6 NADH, 6H+, 2 FADH2
Pentose phosphate pathway and its regulation
Gluconeogenesis, glycogen metabolism
- Gluconeogenesis
3 big difference steps regulate the whole process. These reactions are tightly controlled so that glycolysis and gluconeogenesis are not run at the same time. If they were, the F1,6BP ⇌ F6P reaction could turn into a futile cycle, using up ATP without progressing in either direction.
GTP –> GDP + CO2
F-1,6-BP –> F-6-P (H20 –> Pi) - Glycogen degradation (Glycogen catabolism): Release glucose from glycogen. Triggers glycogen in your liver to convert back to glucose so it can enter your bloodstream.
a. Step 1: The release of glucose - 1- P from glycogen
Enzyme Glycogen phosphorylase catalyses, it cleaves the alpha-1,4-glycosidic linkage by using orthophosphate (HPO4-)
Glycogen + Orthophosphate —> Glucose-1-P + (Glycogen) n-1
b. Step 2: The remodelling of glycogen substrate to permit further degradation
Since glycogen phosphorylase can only cut alpha-1,4-glycosidic bond, more enzymes are used to cut the alpha-1,6-glycosidic bond
- Transferase: transfer 3 glucose residues from 1 branch to another => Leave 1 glucose that linked via alpha-1,6-glycosidic bond
- alpha-1,6-glucosidase: cleaves alpha-1,6-glycosidic bond to release to free glucose and forms a modified glycogen
=> The branched is converted into a linear
c. Step 3: Glucose-1-P to Glucose-6-P by phosphoglucose mutase
d. Step 4: Glucose-6-P to Glucose by glucose-6-phosphatase
The catabolism and anabolism of fatty acids
Beta-oxidation is the catabolic process by which fatty acid molecules are broken down in the mitochondria to generate acetyl-CoA.
Beta-oxidation process: Most of the changes will happen in beta carbon
- Alpha carbon: the carbon attaches to the functional group(CoA functional group is C=O-S-CoA, in this case, it is the 2nd carbon starting counted from the CoA)
- beta carbon: next to alpha cabon
Step 1: Generated a trans-double bond (not fatty acyl !!!!!!!!!!!)
Step 2: Hydration –> Add an OH group into the beta-C atom
Step 4: Ketone –> Activated fatty acyl group + Activated Acetyl group
–> Carbon chain is shorten 2C
Amino acid catabolism and urea cycle
- Amino acid catabolism
Imagine we ate our meal and our body breaks down protein into amino acids. There is a bunch of alpha amino acids, which our body can use to form new proteins. The unused amino acid in our body must be excreted because our body has no way to store it in a long term. So they have to undergo transamination and oxidative deamination to ultimately get rid of these alpha amino acids by our body.
a. Step 1: Transamination (Transfer of amino group to alpha-ketoglutarate to form glutamate)
Amino acid is converted into keto acid, catalyzes by amino transferases. This keto acid then is used for energy to form ATP.
The amino group acceptor is alpha-ketoglutarate ( the universal nitrogen acceptor in our bodies) and itself gets converted into glutamate. We take alpha-ketoglutarate from Krebs cycle but we will regenerate it at the end
Glutamate is the only amino acid that can rapidly undergo oxidative deamination to liberate the nitrogen group in the form of NH3
b. Step 2: Oxidative deamination (Remove of amino group)
Involves the liberation of the amino group in the group of ammonia and the regeneration of alpha-ketoglutarate that we used before.
The enzyme glutamate dehydrogenase can quickly catalyzes the oxidative deamination. It is found throughout the body, but predominantly in our liver and kidney.
NAD+ is reduced to NADH and H+. Glutamate is converted into NH3 and alpha-keto glutarate. - Urea cycle: In liver
Structure and function of DNA and RNA
Storage, flow, expression of genetic information, regulation of gene expression
- Gene expression: The transformation of DNA information into functional molecules (Protein)
DNA stores genetic information, RNA transmites and expresses genetic information by directing the synthesis of thousands of proteins found in living organisms. - DNA replication is the process by which a double-stranded DNA molecule is copied to produce two identical DNA molecules. Replication is an essential process because, whenever a cell divides, the two new daughter cells must contain the same genetic information, or DNA, as the parent cell.
The replication process relies on the fact that each strand of DNA can serve as a template for duplication. DNA replication initiates at specific points, called origins, where the DNA double helix is unwound. A short segment of RNA, called a primer, is then synthesized and acts as a starting point for new DNA synthesis. An enzyme called DNA polymerase next begins replicating the DNA by matching bases to the original strand. Once synthesis is complete, the RNA primers are replaced with DNA, and any gaps between newly synthesized DNA segments are sealed together with enzymes.
DNA replication is a crucial process; therefore, to ensure that mistakes, or mutations, are not introduced, the cell proofreads the newly synthesized DNA. Once the DNA in a cell is replicated, the cell can divide into two cells, each of which has an identical copy of the
original DNA. - Transcriptional regulation – controlling the rate of gene transcription for example by helping or hindering RNA polymerase binding to DNA
Transcriptional regulators - a substance, such as a protein, that bind to specific DNA sequences in order to regulate the expression of a gene
The function of TFs is to regulate—turn on and off—genes in order to make sure that they are expressed in the desired cells at the right time and in the right amount throughout the life of the cell and the organism
Characteristics of restriction enzymes and their use in molecular biology
RE is a protein isolated from bacteria and archaea. Original function in bacteria is prevent the growth of bacteriophage —> Protect the cells by breaking down into smaller non-functional fragments
Example: EcoR I—> produced by E.coli, Taq I —> from Thermus aquaticus
They can also be synthesize artificial: crispr rna molecules
Most RE sites are palindromic: The sequence that is recognized reads the same forwards and backwards
Application:
1. Isolate DNA sequence and make recombinant DNA
2. Determining the nu sequence of long DNA molecules: RE cut the sequence into smaller pieces to recognize the nu seq -> Piecing together and determining the order of fragments —> determine the order of nu in dna
3. Detect SNP ( single nu polymorphism): a genetic disorder at which 1 single nu is replaced with another type of nu in a DNA sequence. It occurs in 1% of the population
. Example: a specific base position of the genome contains a G nu. But the minority of individuals may have the nu A —> 2 possible variations of the nu position are G and A.
5. Cloning and amplifying DNA
Describe the basic characteristics of vectors and their application in molecular biology
Plasmid:
- Can contain genes that produce toxins, give the cell resistance to antibiotics, or produce proteins needed to break down cell products
- Use in lab to manipulate gene expression in target cells.: Produce a large amount of protein so that scientists can purify and study it in a controlled setting, produce enzymes that will make specific, controlled changes to an organism genome …
- Plasmid transfer their genetic materials to other bacteria through 3 mechanism in Horizontal Gene Transfer -> Conjugation provides a quick way for plasmid to spread through a population
Phage: can carry recombinant DNA into a bacterial cell.
How can we use lambda phage as vectors: We replace the DNA original in the phage and replace with our DNA of choice, which has the same size with the original one. Cut the lambda phage DNA with RE —> Remove one fragment and replace it with the DNA of interest, connected by DNA ligase —> Insert the recombinant DNA into the lambda phage, which can be then used to infect bacterial cell
- Actually lambda phage doesn’t need its own DNA to survive, it can use any DNa as long as has pretty much the same size of that original one.
- Cosmid
COS site: needed for packing DNA into the phage particles - Artificial chromosomes: can carry large DNA insert
Theoretical basis and application of PCR
Significance of genome sequences in biological research
All organisms (bacteria, vegetable, mammal) have a unique genetic code, or genome, that is composed of nucleotide bases (A, T, C, and G). If you know the sequence of the bases in an organism, you have identified its unique DNA fingerprint, or pattern. Determining the order of bases is called sequencing.
A genome is the complete set of DNA sequences in an organism and contains all of the instructions required for that organism to function, including embryogenesis, growth, responding to the environment, and healing from disease.
Whole genome sequencing is a laboratory procedure that determines the order of bases in the genome of an organism in one process.
Scientists conduct whole genome sequencing by following these four main steps:
1. DNA shearing: using molecular scissors to cut the DNA, which is into pieces that are small enough for the sequencing machine to read.
–> DNA bar coding: Scientists add small pieces of DNA tags, or bar codes, to identify which piece of sheared DNA belongs to which bacteria.
–> DNA sequencing: The bar-coded DNA from multiple bacteria is combined and put in a DNA sequencer. The sequencer identifies the A’s, C’s, T’s, and G’s, or bases, that make up each bacterial sequence. The sequencer uses the bar code to keep track of which bases belong to which bacteria.
–>Data analysis: use computer analysis tools to compare sequences from multiple bacteria and identify differences. The number of differences can tell the scientists how closely related the bacteria are, and how likely it is that they are part of the same outbreak.
Sequencing an entire genome (all of an organism’s DNA) remains a complex task. It requires breaking the DNA of the genome into many smaller pieces, sequencing the pieces and assembling the sequences into a single long “consensus”.
DNA sequencing is the process of determining the sequence of nucleotide bases in a piece of DNA.
1. Sanger sequencing ingredients: Primer, DNA polymerase, dNTPs, template DNA, ddNTPs (di-dNTP, they lack OH- group on the 3rd carbon) —> Quite similar to those needed for PCR
- Steps: Denaturation —> Annealing —> Elongation, starting from the primer, until it happens to add a dideoxy nucleotide instead of deoxynucleotide —> The strain will end with the ddNTP—> Repeat the cycle, until ddNTP incorporates at every single position of the target DNA in at least 1 reaction. The tube will contain fragments of different length, which ends will be labelled with dyes that indicate their final nucletide —> Run capillary gel electrophoresis —> Chromatogram
- Next gen sequencing: Many sequencing reactions occur at the same time, low cost, fast, …
Examining the function of genes, describe the microarray method
- Gene
Gene, a unit of hereditary information, occupies a fixed location (locus) on a chromosome. It contains the complete instructions for protein production.
- They are made up of Promoter regions, alternating regions of introns (non-coding sequence), exons (coding sequence)
The function of genes:
- Control the function of DNA and RNA
- Code for specific proteins, or segments of proteins, which have different functions within the body. The nucleotide sequence of a gene DNA specifies the amino acid sequence of a protein through genetic code. Sets of 3 nu, known as codon, represents 1 aa
- Contain a particular set of instructions (Globin gene is instructed to produce hemoglobin)
- Genes are passed from parent to offspring and contain the information needed to specify physical and biological traits.
- can acquire mutations in their sequence, leading to different variants, known as alleles, in the population.
- Microarray method: Detect the expression of thousands of genes
- DNA microarrays are microscope slides that are printed with thousands of tiny spots in defined positions. Each spot contains a known single-stranded DNA sequence or gene, which is called “probe”. The DNA microarray contains many or even all of the genes of an organism, called “gene chips”
Application of microarray method:
- Analyse a larger number of samples which have either been recorded previously or new samples
- Measure gene expression: Determine which genes are activated or repressed when 2 populations of cells are compared
- Observe mutation in DNA: Compare gene expression in a regular cell and a cancer cell in human body.
Steps of microarray methods:
1. Sample collection:
Two types of samples are collected, i.e. healthy and infected cells, for comparing and obtaining the results.
- Isolation of mRNA:
- The extraction of RNA from a sample is performed by using a column or solvent like phenol-chloroform.
- mRNA is isolated from the extracted RNA leaving behind rRNA and tRNA, or by transcription of DNA
- As mRNA has a poly-A tail, column beads with poly-T tails are employed to bind mRNA.
- Buffer is used to rinsing the column in order to isolate mRNA from the beads. - Creation of labeled cDNA:
- Reverse transcription of mRNA yields cDNA. (cDNA has the sequence that identical to segment of genes that produce mRNA because DNA is the template for mRNA under transcription)
- Both the samples are then integrated with different fluorescent dyes for the production of fluorescent cDNA strands which allows to differentiate the sample category of the cDNAs. - Hybridization:
- The labeled cDNAs combined from both samples are put into a single tube, then they are placed on the DNA microarray which permits the hybridization of each cDNA to any DNA complementary strand they can find. Complementary base pairing between cDNA and the sample DNA occurs to detect gene expression. (Which color is seen in the microarray, means that gene is expressed in that tissue). Binding means gene is expressed.
- Then they are thoroughly washed to remove unpaired sequences. - Collection and analysis:
Microarray scanner is used to collect the data.
The scanner contains a laser, a computer and a camera. The laser is responsible for exciting the fluorescence of the cDNA, generating signals.
(The camera records the images produced at the time laser scans the array.
Then computer stores the data and yields results instantly.) The data are now analyzed.
The distinct intensity of the colors for each spot determines the character of the gene in that particular spot.
For example, cDNAs in sample 1 are labelled red, sample 2 are green
—>Red spot means gene is expressed at sample 1
Green spot means gene is expressed at sample 2
Yellow spot means gene is expressed in both sample
No color means no gene expression
