Biomidterm II - Nov 11th Flashcards
Describe myoglobin
- Only in cardiac myocytes and skeletal muscle fibers of vertebrates
- The major function of myoglobin is to facilitate
oxygen diffusion in muscles and act as oxygen storage - Myoglobin is not essential for muscles under normal conditions
Describe the shape / size of myoglobin
- Contain 153 amino acids (sperm whale myoglobin). First protein with known X-ray structure.
- Human myoglobin contains 154 amino acids
- Contain 8 helices: A-H and short inter helices region such as CD, EF and GH
Describe the heme group of myoglobin
- Heme is a porphyrin derivative
containing four pyrrole groups
(Proline side chain is a
pyrrolidine group) - Heme occurs in many proteins:
myoglobin, hemoglobin,
neuroglobin, cytochrome c etc - Heme sits in a hydrophobic
pocket - Oxygenation alters the
electronic state of the Fe2+-
Heme complex and causes the
color change
What helps hold heme in place in the myoglobin?
- Val E11 and Phe CD1 stabilise the heme group: The amino acids Valine (Val E11) and Phenylalanine (Phe CD1) create a hydrophobic pocket that stabilises the heme within myoglobin, ensuring it stays embedded in the protein and can bind oxygen effectively.
How do you calculate for oxygen binding property of myoglobin?
How do you calculate for oxygen binding property of myoglobin as a function of partial pressure
Describe the oxygen binding curve of myoglobin
- As more oxygen gets dissolved in the blood (pO2 increases) more myoglobin are saturated (YO2 increases)
- pO2 = the amount of total oxygen in the blood that is dissolved
- k = the partial pressure of oxygen when 50% of myoglobin are saturated (have oxygen bound to their heme group)
Describe the function of hemoglobin
*Located in red blood cells
* Structurally (homologue) related to myoglobin
*But only 18% of residues are identical in myoglobin and in the alpha or beta subunits of hemoglobin
*Functions for O2 transport
Describe hemoglobin as a tetramer and its symmetry
In C₂ symmetry, there is a single axis of symmetry around which the structure can be rotated by 180 degrees to yield an identical configuration
In a binding affinity curve for hemoglobin and myoglobin:
- _____ binding curve
always shows a cooperative
binding: binding of one
ligand affects the other
ligand binding sites - _____ binding curve
always shows an
independent binding:
binding of one ligand does
not affect the other ligand
binding sites
Sigmoidal
Hyperbola
Describe the Hill equation
Describe how the Hill plot tells how hemoglobin binds the first
and the next O2 so differently
Why does hemoglobin have such a binding property?
( lower O2 concentrations, low O2 binding affinity, at higher O2
concentrations, higher O2 binding affinity )
Structure and Subunit Interaction: Haemoglobin is made up of four subunits, each containing a heme group that can bind one O₂ molecule. When one O₂ molecule binds to one of these heme groups, it causes a conformational (shape) change in the haemoglobin molecule. This structural shift increases the affinity of the remaining subunits for O₂.
Describe the T and R state of hemoglobin
Haemoglobin shifts between two key structural states to manage its oxygen-binding properties:
T State (Tense State):
- This is the low-affinity form, where haemoglobin binds oxygen weakly. It’s stabilised by salt bridges and hydrogen bonds, which create a “tense” structure resistant to oxygen binding.
- The T state predominates in low-oxygen areas like body tissues, facilitating oxygen release to meet tissue demand.
R State (Relaxed State):
- In the high-affinity R state, haemoglobin binds oxygen more readily. When one oxygen molecule binds to a haemoglobin subunit in the T state, structural changes break salt bridges, shifting the entire molecule into the R state.
- This conformation is favoured in high-oxygen environments like the lungs, where haemoglobin can fully load with oxygen for transport.
Describe the role of porphyrin in hemoglobin
- Structure: Porphyrin is a ring-shaped molecule made of four pyrrole units, holding an iron (Fe²⁺) atom at its centre.
- Oxygen Binding: The iron atom binds oxygen reversibly, allowing haemoglobin to transport oxygen in the blood.
- Stabilisation: Porphyrin stabilises the iron in its Fe²⁺ state, preventing oxidation to Fe³⁺, which cannot bind oxygen.
- Conformational Changes: When oxygen binds to iron, it induces a structural shift in haemoglobin, facilitating the transition between the T (tense) and R (relaxed) states and regulating oxygen affinity.
Describe the Changes at the alpha1-beta2 interface during
the T–>R transition in hemoglobin
How do both Both alpha and beta chains C-termini form
ion pairs
(Arg 141alpha and His 146beta)
Describe the Bohr effect
The Bohr effect is a physiological phenomenon where an increase in carbon dioxide (CO₂) concentration and a decrease in pH (increased acidity) lead to a reduction in hemoglobin’s affinity for oxygen. This effect facilitates oxygen release in metabolically active tissues, where CO₂ production and acidity are high. As a result, hemoglobin delivers more oxygen to tissues that need it most, enhancing overall oxygen transport efficiency in the body.
What is the role of Bisphosphoglycerate in hemoglobin?
BPG Stabilises the T State: 2,3-bisphosphoglycerate (BPG) binds tightly to the T state of hemoglobin, occupying a specific channel and stabilising this low-affinity conformation, which shifts the equilibrium away from the high-affinity R state.
Narrowing of the Binding Channel: The binding channel for BPG in hemoglobin is much wider in the T state, allowing for effective binding, while it becomes narrower in the R state, making it difficult for BPG to bind and facilitating higher oxygen affinity.
Enhanced Oxygen Release: By stabilising the T state, BPG promotes the release of oxygen in tissues with high metabolic activity, where oxygen demand is greater, ensuring efficient oxygen delivery tailored to the physiological needs of the body.
How are O2 and CO2 transport through the blood
How many ways can CO2 be transported?
- About 7-10% of CO₂ is transported dissolved in plasma, where it remains in its molecular form.
- Approximately 20-25% of CO₂ binds to hemoglobin and other proteins, forming carbamino compounds that facilitate its transport.
- The majority, around 70-75%, is converted into bicarbonate ions through a reaction with water, allowing for efficient transport in the plasma
Describe Mutations that Alter Hb’s Structure & Function
Is there a significant overlap in between areas with sickle cell anemia and malaria? and why?
Yes, there is a significant overlap between areas affected by sickle cell anemia and malaria, particularly in sub-Saharan Africa and parts of India and the Mediterranean. This overlap exists because the sickle cell trait provides a protective advantage against malaria; individuals with the trait are less likely to suffer severe forms of the disease. As a result, the prevalence of the sickle cell trait has increased in regions where malaria is endemic, illustrating how genetic traits can evolve in response to environmental pressures.
- How many alpha-helices are there in a myoglobin protein?
- What ion does the myoglobin protein bind?
- Which amino acid holds the metal ion in the myoglobin?
- Number of α-Helices: Myoglobin contains eight α-helices (labeled A through H) that form its compact structure.
- Ion Bound by Myoglobin: Myoglobin binds an iron (Fe²⁺) ion, which is crucial for its function in oxygen binding.
- Amino Acid Holding the Metal Ion: The metal ion (iron) in myoglobin is held in place by a histidine residue, specifically the proximal histidine (His93), which coordinates directly to the iron atom in the heme group.
When the partial pressure of oxygen in venous blood is
30 torr, the YO2 value for myoglobin is ______ given
that the k value is 2.8 torr.
a) 0.55
b) 0.91
c) 2.8 torr
d) 0.95
e) none of the above
Describe carbohydrates
Describe the function of carboydrates
❑Mainly act as energy resource
❑Protection as structural materials
❑Intercellular communication
❑Stabilization of protein structures
Describe monosaccharides
Describe Aldose (containing 3 to 6 carbon atoms)
Describe Ketose (containing 3 to 6 carbon atoms)
Describe Cyclic monosaccharide structures and anomeric forms
Describe how ketose relates to D-enantiomers and epimers
Describe how Howarth projections represent sugars
Haworth projections are a two-dimensional representation of cyclic monosaccharides that effectively convey stereochemical information.
- Cyclic Structure: They depict sugars in their cyclic forms (pyranose or furanose) as flat rings, with each vertex representing a carbon atom.
- Anomeric Carbon: The anomeric carbon is clearly marked, with the orientation of the hydroxyl group indicating whether it is in the α (downward) or β (upward) configuration.
- Substituent Orientation: Other substituent groups are shown with their positions indicating stereochemistry, and hydrogen atoms are often omitted for simplicity.
Describe the Two anomeric forms of cyclic sugars
The two anomeric forms of cyclic sugars are alpha (α) and beta (β), distinguished by the orientation of the hydroxyl group (-OH) on the anomeric carbon:
- Alpha (α) Anomer: The -OH group on the anomeric carbon is positioned downward (trans to the CH₂OH group) in the Haworth projection. For example, α-D-glucose features this configuration.
- Beta (β) Anomer: The -OH group is positioned upward (cis to the CH₂OH group), as seen in β-D-glucose.
- These forms can interconvert in solution through mutarotation, where the cyclic structure temporarily opens to allow the hydroxyl group to change orientation, impacting the sugar’s properties and biological functions.
Are monosaccharides in planar confirmation?
Describe how sugars are modified to produce aldonic and uronic acids
Describe how sugars are modified to produce alditols and deoxy sugars
Describe sugar acetylation
Describe sugar phosphorylation
Describe N-Acetylneuraminic for complex modification of sugars
- N-Acetylneuraminic acid (Neu5Ac) is a nine-carbon sugar, also known as sialic acid, that plays a vital role in the modification of glycoconjugates like glycoproteins and glycolipids.
- Structure and Composition: It contains an N-acetyl group and is characterized by its cyclic form, which includes a carboxyl group, a hydroxyl group, and an acetamido group.
- Biological Significance: Neu5Ac is crucial for cell-cell recognition, immune response modulation, and neurological functions, often found at the terminal positions of glycan chains, enhancing cell adhesion and preventing immune detection.
- Complex Sugar Modification: Through the process of sialylation, Neu5Ac modifies glycoproteins and glycolipids, impacting their stability, biological activity, and overall functionality, contributing to a diverse range of glycan structures with specific biological roles
Describe how the anomeric group of a sugar can condense
with an alcohol to form alpha or beta-glycoside
Describe N-glycosidic bonds
Found in:
- Nucleotides: In DNA and RNA, an N-glycosidic bond connects the nitrogenous base (adenine, thymine, cytosine, guanine, or uracil) to the 1’ carbon of the sugar (ribose in RNA or deoxyribose in DNA).
- Glycoproteins: N-glycosidic bonds link sugars to the amino group of asparagine residues in glycoproteins, forming N-linked glycosylation.
Describe cellulose
Describe chitin
What is the main storage polysaccharide in plants?
What is the main storage polysaccharide in animals?
Glycoconjugates are molecules that consist of carbohydrates covalently bonded to other types of biomolecules, such as proteins or lipids. This combination allows glycoconjugates to play essential roles in cell recognition, signalling, and structural integrity. Key types and roles include:
Proteoglycans: These are heavily glycosylated proteins, containing large carbohydrate (glycosaminoglycan) chains. Proteoglycans are abundant in connective tissue, providing structural support and participating in cellular signalling pathways
Describe Peptidoglycans
Bacterial cell walls
What are the two types of bacterial cell walls?
Describe NAG and NAM
N-Acetylglucosamine (NAG): This amino sugar has a six-carbon structure with an acetyl group at the amino position. It forms part of the repeating disaccharide units in peptidoglycan, linking to NAM through β-1,4-glycosidic bonds.
N-Acetylmuramic Acid (NAM): Similar to NAG but contains a lactic acid side chain at the C3 position, NAM also alternates with NAG in the peptidoglycan structure. Its unique side chain allows for peptide cross-linking, enhancing the rigidity and strength of the bacterial cell wall.
Describe pentaglycine
Structure and Function: Pentaglycine provides flexibility and connectivity in the peptidoglycan layer, contributing to its structural integrity and rigidity.
Linkage with Lysine and D-Alanine: Pentaglycine links the side chain of a lysine (Lys) residue from one tetrapeptide to the D-alanine (D-Ala) of a neighboring tetrapeptide, forming covalent cross-links.
Cross-Linking Importance: This interaction enhances the strength and resistance of the peptidoglycan structure, which is essential for bacterial survival against osmotic pressure.
Describe penicillin as and antiobiotic and beta-lectamase as an antagonist
Penicillin is a beta-lactam antibiotic that inhibits bacterial cell wall synthesis by targeting penicillin-binding proteins (PBPs), which are essential for cross-linking peptidoglycan layers.
Beta-Lactamase Resistance: Some bacteria produce beta-lactamase enzymes that hydrolyze the beta-lactam ring in penicillin, rendering it ineffective.
Combination Therapy: To combat this resistance, penicillin is often combined with beta-lactamase inhibitors, such as clavulanic acid, which protect penicillin from degradation, enhancing its effectiveness against resistant bacteria.
This combination therapy improves the treatment of infections caused by beta-lactamase-producing bacteria, maintaining the utility of penicillin in clinical settings.
Describe glycoproteins
What are the two types of glycoproteins
- O-linked glycoproteins: carbohydrate is linked to the protein through OH group the side chain of serine, threonine or tyrosine
- N-linked glycoproteins: carbohydrate is linked to the protein through the side chain of Asparagine (N)
Describe Glycoprotein biosynthesis in Eukaryotic cells
Glycoprotein biosynthesis in eukaryotic cells involves several key steps:
- Protein Synthesis: The process begins with the transcription of the gene encoding the glycoprotein into mRNA, followed by translation into a polypeptide chain in the cytoplasm. A signal peptide directs the nascent protein to the endoplasmic reticulum (ER).
- Glycosylation: In the ER, the polypeptide undergoes co-translational translocation and folding, followed by N-glycosylation, where an oligosaccharide is attached to asparagine residues. O-glycosylation occurs in the Golgi apparatus, adding sugars to serine or threonine residues.
- Golgi Modifications and Transport: The glycoprotein is transported to the Golgi apparatus, where it undergoes further modifications and sorting for its final destination, such as the plasma membrane or secretion outside the cell.
Describe the most common form of O-linked glycoproteins
O-linked carbohydrates can include any single or short carbohydrate group linked via oxygen.
O-linked oligosaccharides specifically refer to more complex, branched sugar chains attached through oxygen, often with intricate biological roles.
Describe N-linked glycoproteins
Describe N-linked Protein Glycosylation In The ER
Describe the Biosynthesis Of N-linked Glycoproteins
Describe N-linked Glycosylation In ER And Golgi
Diagram
Describe lipid functions
Describe the four classes of lipids
Describe fatty acids
Compare Saturated vs. Unsaturated Fatty Acids
When naming common biological fatty acids, if the acid is listed as 18:3n-3 what does that mean?
- The 18 = total number of carbons in the acid
- The 3 = number of double bonds
- The second 3 = position of last double bond from terminal methyl group
Describe hydrogenation of unsaturated fats
Hydrogenation Process: Hydrogenation is the chemical process of adding hydrogen to unsaturated fats, often converting oils into a more solid form. This process is typically achieved by exposing the fat to hydrogen gas at high temperatures in the presence of a metal catalyst (usually nickel).
Formation of Trans Fats: Partial hydrogenation can cause some of the natural cis configurations of double bonds in unsaturated fats to flip into trans configurations. This creates trans fats, which have a straighter molecular shape, allowing them to solidify at room temperature.
Health Implications: Trans fats are associated with negative health effects, as they can raise ____ (bad) cholesterol and lower ____ (good) cholesterol, increasing the risk of cardiovascular diseases.
LDL
HDL
Describe Triacylglycerols
Structure: Triacylglycerols (also called triglycerides) are lipid molecules consisting of three fatty acid chains attached to a glycerol backbone. Each fatty acid is linked to glycerol through an ester bond, forming a stable structure.
Function: Triacylglycerols serve as the body’s primary form of long-term energy storage. They are stored in adipose tissue and can be broken down during fasting or energy-demanding activities to release fatty acids and glycerol for fuel.
Properties: Triacylglycerols can be solid (fats) or liquid (oils) at room temperature, depending on the types of fatty acids they contain. Saturated fats are more likely to be solid, while unsaturated fats are usually liquid.
Describe Glycerophospholipids
Structure: Glycerophospholipids are composed of a glycerol backbone, two fatty acid chains, and a phosphate group that is often further modified with additional polar head groups (such as choline, ethanolamine, or serine). This amphipathic structure allows them to form bilayers in aqueous environments.
Function: They are essential components of cell membranes, contributing to membrane fluidity and integrity. The hydrophobic tails face inward, while the hydrophilic head groups face outward, creating a barrier that separates the internal and external environments of the cell.
What are the common classes of glycerophospholipids (based on their polar head group)
What are Sphingolipids and how are they important for higher animals?
Cell Membrane Structure and Fluidity: Sphingolipids, including gangliosides and ceramides, are essential for maintaining cell membrane integrity and fluidity. Their unique structures contribute to the formation of lipid rafts, which support various membrane proteins and signalling pathways, particularly in neurons.
Cell-Cell Recognition and Communication: Gangliosides play a crucial role in cell-cell interactions, mediating immune responses and neuronal communication. They serve as recognition molecules that influence how cells respond to pathogens and communicate during tissue development and repair.
Regulation of Cellular Processes: Ceramides are important for regulating processes such as apoptosis and inflammation. They act as signalling molecules that can trigger programmed cell death, crucial for tissue homeostasis, and are involved in inflammatory responses linked to diseases like obesity and neurodegeneration
Describe Sphingolipids
Structure: Sphingolipids are composed of a sphingosine backbone, a long-chain fatty acid, and a polar head group. Unlike glycerol-based lipids, sphingolipids contain a single fatty acid linked to the sphingosine through an amide bond, which gives them unique properties.
Function: They play critical roles in cellular signalling and recognition. Sphingolipids are essential components of cell membranes, particularly in the nervous system, where they contribute to the formation of myelin sheaths that insulate nerve fibres.
Types: Key subclasses of sphingolipids include ceramides, sphingomyelins (which contain a phosphocholine head group), and glycosphingolipids (which have sugar residues).
Describe cholesterol
Describe Vitamin D2 & D3 as Sterol Derivatives
Chemical Structure: Vitamin D2 (ergocalciferol) and D3 (cholecalciferol) are sterol derivatives derived from cholesterol. D2 is produced by UV irradiation of ergosterol, while D3 is synthesised in the skin upon UVB exposure.
Biological Functions: Both vitamins are essential for calcium and phosphorus metabolism, promoting bone health by enhancing the absorption of these minerals in the intestine. They regulate parathyroid hormone levels, support bone mineralisation, and help maintain bone density. Additionally, Vitamin D plays a role in immune function, influencing the activity of immune cells and reducing inflammation, which may have implications for chronic disease prevention and overall health
Just look at this
Why are glycerophospholipid and sphingolipid the major membrane components?
Amphipathic Nature: Glycerophospholipids and sphingolipids have an amphipathic structure, with hydrophobic tails and hydrophilic head groups. This allows them to form stable bilayers in aqueous environments, creating a barrier that separates the cell’s interior from the outside.
Membrane Fluidity: These lipids enhance the fluidity and flexibility of cell membranes. Glycerophospholipids can vary in fatty acid composition, while sphingolipids help organise membrane proteins, which is essential for proper cellular function.
Cell Signalling: Both types of lipids are involved in cell signalling and recognition. Glycerophospholipids participate in signalling pathways, and sphingolipids, such as gangliosides, facilitate cell-cell communication, impacting processes like growth and immune responses.
Membrane lipids are divided ____ between the inner and outer leaflet
Describe the inner v.s outer leaflet
Composition Differences: The inner and outer leaflets of the plasma membrane differ in their lipid and protein compositions. The outer leaflet predominantly contains sphingolipids and phosphatidylcholine, which contribute to a positive charge on the extracellular side. In contrast, the inner leaflet is rich in phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, which play key roles in intracellular signalling and membrane dynamics.
Functional Roles: The distinct compositions of the leaflets contribute to their specific functions. The outer leaflet is involved in cell recognition, signalling, and interactions with the extracellular environment, while the inner leaflet is crucial for anchoring cytoskeletal proteins and facilitating intracellular communication.
Asymmetry Importance: The asymmetry between the inner and outer leaflets is vital for maintaining membrane integrity and functionality. This asymmetry affects membrane fluidity, stability, and the ability to respond to external signals, ensuring proper cellular processes such as apoptosis and vesicle trafficking
Describe fluid-like lipid bilayers
Describe glycans
= Glycans are complex carbohydrates composed of sugar molecules that are linked together to form larger structures, such as oligosaccharides and polysaccharides. They play crucial roles in biological processes, including cell recognition, signalling, and the formation of structural components in cells and tissues.
What are the three types of membrane proteins
What are the four classes of membrane anchored proteins
Describe Prenylated proteins
Describe Palmitoylated proteins
Describe Myristoylated proteins
Myristoylated proteins are proteins that have a myristoyl group (a 14-carbon saturated fatty acid) covalently attached to their N-terminal glycine residue, which helps anchor them to cell membranes. This lipid modification facilitates membrane association, influencing protein localization, stability, and interactions in various cellular processes, including signal transduction.
Describe GPI-anchored proteins
Describe integral proteins
Describe the three types of transmembrane proteins
Describe Transmembrane Segments that Contain
Either alpha-helices or beta-barrels
Describe beta-barrel proteins
Describe bacteriorhodopsin
Describe glycophorin A
Describe the topology of membrane proteins
Describe the Properties of Membrane Proteins
(alpha-helix TM Segment)
Where does N-Glycosylation occur (specific, include location parameters for the protein sequence)
Describe the roles of signal recognition particles and signal peptides in polypeptide synthesis
Describe signal peptide cleavage
Describe translocon
- Translocon embedding of TM proteins happens in four steps:
1. Signal peptide on end of chain attaches to wall of translocon
2. Protein (peptide chain) is fed through translocon until the hydrophobic stop transfer signal halts production
3. Signal peptidase cleaves the signal sequence
4. Peptide sequence moves laterally through the translocon and embeds itself into the membrane
What are the three types of vesicles involved in intracellular transport
Describe membrane fusion phase of vesicle
How do SNARE proteins participate in membrane fusion
Does the lipid membrane allow free diffusion of most substances?
What are the two types of membrane transport
Describe the two ionophores involved in passive transport
How do you calculate the electrochemical potential
Describe Kcs A
What are the five types of ATPases / transporters required for active transport
- F-Type = ATP synthase (pump proton, make ATP)
- V-Type = Pump proton into lysosomes, endosome, (for degredation reasons?) use ATP
- A-Type = found in archaea and work like F-types but pump anions instead of protons
- ABD = Legit just pinball arms that throw molecules from one side to another
Describe Na/K ATPases
In the Na⁺/K⁺ ATPase cycle, E2-P * 2K⁺ represents a specific conformational state of the enzyme. Here’s what it means:
E2: The enzyme is in a conformation that favours the binding and release of potassium (K⁺) ions on the extracellular side of the membrane.
P: Indicates phosphorylation of the enzyme, providing energy for ion transport.
* 2K⁺: Two potassium ions are bound to the enzyme in this state, ready to be transported into the cell.
This step is part of the mechanism allowing the Na⁺/K⁺ pump to maintain ion gradients across the cell membrane.
Describe the six steps involved in the active transport done by Na/K ATPases
Describe uniporters, symporters and antiporters
Tell me what each digit means in the classification number below:
Tripsin: EC 3.4.21.4
Describe oxidoreductases
Describe transferases
Describe hydrolases
Describe lyases
Describe isomerases
Describe the general properties of enzymes
What makes enzyme catalysis highly specific?
Describe stereospecificity in enzymes
Describe chymotrypsin
Chymotrypsin is a digestive enzyme that plays a crucial role in the breakdown of proteins in the small intestine. It is a type of serine protease, meaning it cleaves peptide bonds in proteins using a serine residue in its active site.
Chymotrypsin specifically targets peptide bonds adjacent to large, aromatic amino acids such as phenylalanine, tryptophan, and tyrosine. By breaking these peptide bonds, it helps to degrade proteins into smaller peptides, which are then further broken down into amino acids by other enzymes. This process is essential for protein digestion and the absorption of amino acids, which are used for various physiological functions, including tissue repair and enzyme synthesis.
Describe enzymatic cofactors
What are the 5 mechanisms of catalysis
Acid-Base Catalysis: Enzymes can facilitate reactions by donating or accepting protons (H⁺). Acidic or basic residues in the enzyme’s active site can act as proton donors or acceptors, stabilising transition states or intermediates and speeding up the reaction.
Covalent Catalysis: In this mechanism, the enzyme forms a transient covalent bond with the substrate during the reaction. This intermediate can then be more easily converted to the product. The enzyme often acts as a nucleophile, attacking electrophilic centres of the substrate.
Metal Ion Catalysis: Many enzymes use metal ions to assist in the catalytic process. Metal ions can stabilise negative charges, facilitate electron transfers, or help in substrate binding. Transition metals like Zn²⁺, Fe²⁺, and Mg²⁺ are commonly involved in these reactions.
Proximity and Orientation Effects: Enzymes bring substrates into close proximity and orient them in an optimal position for the reaction to occur. This reduces the entropic barrier to the reaction and increases the probability of effective collisions between the reacting molecules.
Preferential Binding of Transition State Complex: Enzymes bind to the transition state of the reaction more strongly than to the substrate or product. This preferential binding stabilises the transition state, lowers the activation energy, and accelerates the reaction
Describe keto-enol tautomerisation
Describe RNase A as an acid-base catalyst.
And how many S-S in RNase A ?
- RNase A is an enzyme that acts as an acid-base catalyst in the cleavage of RNA molecules. It catalyzes the hydrolysis of the phosphodiester bond in RNA through a two-step mechanism, where His12 and His119 act as key acid-base catalysts. These histidine residues alternately donate and accept protons to facilitate the reaction, helping to stabilize the transition state.
- As for the number of S-S bonds in RNase A, there are two disulfide bonds (S-S) in its structure. These bonds help maintain the enzyme’s stable three-dimensional conformation, which is essential for its catalytic activity.
Describe the phosphate ester break in RNase A
Think His
Describe covalent catalysis
Covalent catalysis: This involves the formation of a temporary covalent bond between the enzyme and the substrate. The enzyme acts as a nucleophile and forms a covalent intermediate, which lowers the activation energy for the reaction. The enzyme then helps to break the bond and release the product.
What are the three stages of covalent catalysis
- Nucleophilic reaction between the catalyst and the substrate to form a covalent bond
- Withdrawal of electrons from the reaction
center by the now electrophilic catalyst - Elimination of the catalyst, a reaction that is essentially the reverse of stage 1
Describe the decarboxylation of acetoacetate
Formation of a carbanion: The decarboxylation of acetoacetate starts with the formation of a carbanion at the carbon adjacent to the carbonyl group (α-carbon). This occurs through the deprotonation of this carbon by a base or through an internal rearrangement.
Elimination of CO₂: The carbanion formed on the α-carbon is highly unstable and quickly breaks down by the elimination of a carbon dioxide (CO₂) molecule.
Formation of acetone: After the elimination of CO₂, the remaining intermediate rearranges to form acetone (CH₃COCH₃), a stable ketone.
Describe metal ion cofactors that act as catalysts for carbonic anhydrase
In carbonic anhydrase, the residues His94, His119, His96, Thr199, and Glu106 are critical for metal-ion catalysis involving zinc (Zn²⁺). His94, His119, and His96 coordinate the zinc ion, enabling the activation of water molecules to form hydroxide ions, which then nucleophilically attack CO₂ to form bicarbonate. Thr199 stabilises the active site and aids in positioning the substrate, while Glu106 helps deprotonate water molecules and stabilise the transition state. Together, these residues ensure efficient catalysis of the CO₂ to bicarbonate reaction.
What are the two types of enzymes containing metal ions
How can catalysis occur through proximity and orientation effects?
Proximity and orientation effects in enzyme catalysis involve bringing substrates together and positioning them in the correct orientation within the enzyme’s active site. While simply concentrating substrates can slightly speed up a reaction (up to five times), the real catalytic benefit comes from the enzyme’s ability to properly orient the substrates. This alignment minimizes entropy and allows for more efficient bond formation or breaking, greatly reducing activation energy. By restricting substrate motion and ensuring optimal orientation, enzymes can accelerate reactions by up to 10^7 times, making them much more efficient than in solution.
Describe the purpose of proline racemase and its significance
Proline racemase catalyses the interconversion of D- and L-proline. It employs two catalytic cysteine residues, which act as general acid-base catalysts, abstracting and donating protons to form a planar carbanion intermediate, the reaction’s transition state. Proline racemase binds the transition state more tightly than the substrate (starting molecule) or the product (resulting molecule). This preferential binding enhances catalytic efficiency because it lowers the energy barrier of the reaction.
Describe the catalytic mechanism of lysozyme
Note: the first 18 codons of chicken lysozyme mRNA are significant as they encode the signal peptide, a short amino acid sequence critical for the protein’s targeting and secretion. Signal peptides are present in many secretory and membrane proteins, and they play a crucial role in directing the nascent protein to the endoplasmic reticulum (ER) for proper processing and transport.
How is lysozyme involved in bacterial cell walls
Lysozymes help break down the beta1-4 bonds of piptidoglycans (alternating NAGs and NAMs) which are key components of the bacterial cell wall. This results in the degredation of the bacteria
Describe lysozyme surfaces
What side chains are involved in substrate binding of lysozymes
What are the five steps of lysozyme catalysis via a covalent intermediate
How do you determine the polysaccharide binding sites on lysozyme
Think Nag and Nam
What is the structure of (NAG)4 when complexed with lysozyme
In the context of the complex with lysozyme, the half-chair structure of the D-ring is crucial for the enzyme’s ability to cleave the glycosidic bond in the polysaccharide. The half-chair form of the ring is a transition-state-like structure that helps facilitate the hydrolysis reaction, as it allows for better positioning of the substrate in the active site and promotes the optimal orientation for bond cleavage. The crystal structure provides direct evidence that lysozyme’s active site stabilizes this conformation, enabling the enzyme to effectively catalyze the breakdown of the (NAG)₄ substrate.
How does mass spectrometry confirm covalent intermediates of lysozyme-NAG
How did they confirm Glu35 and Asp52 as the catalytic sites?
- Da = Dalton = molecular mass in g/mol
- Role of E35 (Glutamate-35):
- Glu35 acts as a general acid during catalysis, donating a proton to facilitate the cleavage of the β-(1,4)-glycosidic bond between NAG and NAM.
- Substituting Glu35 with glutamine eliminates the carboxylic acid group, rendering it unable to act as a proton donor.
- Role of D52 (Aspartate-52):
- Asp52 serves as a nucleophile, forming a transient covalent intermediate with the substrate during the reaction.
*Replacing Asp52 with asparagine removes the negatively charged carboxylate group, preventing nucleophilic attack.
How does the lysozyme bond cleavage get confirmed by isotope labelling
The hydrolytic cleavage of the glycosidic bond by lysozyme is confirmed using isotope labelling with O18-enriched water. During the reaction, the O18 from water is incorporated into the reducing sugar product at its anomeric carbon. Analysis of the product using mass spectrometry or NMR spectroscopy detects the
O18 label, providing direct evidence that water participates in the reaction mechanism. This confirms that lysozyme catalyses glycosidic bond hydrolysis and supports the proposed reaction pathway, including the formation of a covalent intermediate in some cases.
Describe serine proteases
What is the role of Ser and His residues in chemical labelling (think DIPF and S195)
Serine Residues and DIPF (Diisopropylphosphofluoridate):
- Ser195 in enzymes like chymotrypsin is a nucleophilic residue in the active site.
DIPF reacts specifically with the hydroxyl group (-OH) of serine residues in the active site to form a stable phosphorylated derivative, thereby inactivating the enzyme.
Histidine Residues and Specific Labelling:
- His57 in serine proteases works alongside Ser195, often as a general base that activates the serine residue by abstracting a proton from its hydroxyl group.
- Chemical probes such as N-bromoacetyl derivatives or inhibitors can label histidine residues specifically, helping identify their role in the catalytic mechanism.
How were Ser and His residues identified by chemicals labeling
Think TLCK and TPCK
The use of TLCK (trypsin) and TPCK (chemotrypsin) to label histidine residues and DIPF to label serine residues provided crucial evidence for the catalytic triad mechanism in serine proteases (Ser195, His57, and Asp102). These experiments showed how these residues work in concert to facilitate substrate cleavage, with His57 playing a role in proton transfer and Ser195 acting as the primary nucleophile.
Since His57 bidning = inactivation, His must be critical for enzyme work
What is the catalytic triad in trypsin
Ser195, Asp102, His57
Describe the substrate bidning pockets of the three serine proteases
What are the five steps in the mechanisms of serine proteases
Describe oxyanion holes of chymotrypsin
The oxyanion hole stabilises the negatively charged oxygen atom that forms during the tetrahedral transition state in serine proteases. This stabilisation is essential for lowering the activation energy of the reaction, thereby enhancing the efficiency of peptide bond cleavage. The oxyanion hole typically involves hydrogen bonding with the oxygen of the tetrahedral intermediate, ensuring the reaction proceeds smoothly and rapidly.
How was the first tetrahedral state of trypsin directly observed
How was the second tetrahedral state directly observed in crystal trypsin structures
How are proteases synthesized?
Describe reaction coordinate diagrams
Describe enzyme kinematics
What equation explains how enzymes catalyze reaction by preferentially
binding transition state
v: Reaction rate or rate constant.
k: A proportionality constant, often related to the frequency of molecular collisions and orientations.
ΔG‡: The Gibbs free energy of activation, which is the energy barrier that must be overcome for the reaction to proceed.
R: The universal gas constant (8.314 J mol-1K-1)
T: Absolute temperature (in Kelvin).
e: The base of the natural logarithm (≈2.718).
What are the properties of first order reactions and their half-life
The slope of the line = k
Halflife (t1/2) = 0.693/k
What are the properties of second order half life reactions
Describe the early days of biological catalyst research
Describe the Michaelis-Menten equation
What did George Briggs and John Haldane assume about [ES]
What is the revised Michaelis-Menten equation proposed by Briggs and Haldane
Describe typical Michaelis-Menten enzymes
Typical Michaelis–Menten enzymes follow classical kinetics, where reaction rates (v) depend on substrate concentration ([S]) and exhibit a hyperbolic curve. Their key parameters are
Vmax , the maximum reaction rate, and Km , which reflects substrate affinity. These enzymes operate via simple substrate-enzyme interactions without cooperative or allosteric effects, exemplified by enzymes like chymotrypsin and carbonic anhydrase.
Describe the Significance of Michaelis constant (Km)
The Michaelis constant (Km) is a key parameter in enzyme kinetics that represents the substrate concentration at which an enzyme achieves half of its maximum reaction velocity (Vmax). It provides insight into the enzyme’s affinity for its substrate:
- A low Km indicates high substrate affinity, as the enzyme reaches half Vmax at low substrate concentrations.
- A high Km indicates low substrate affinity, requiring higher substrate concentrations to achieve the same reaction rate.
- Km is significant in understanding enzyme efficiency and behaviour under physiological conditions, helping to predict how enzymes function within metabolic pathways and respond to changes in substrate availability.
Describe the catalytic constant (Kcat). How is Kcat/Km is a measure of the catalytic
efficiency
Describe Km, kcat and kcat/Km of example enzymes
Describe the lineweaver-burk equation
Can the kinematic data of enzyme reactions reliably convey the reaction mechanism
Describe common bisubstrate reactions
Describe sequential bisubstrate reactions
Describe ping-pong bisubstrate reactions
What are the two models of competitive inhibition
Describe classical competitive inhibition
What is the significance of competitive inhibitors
Where do the lines cross on a double reciprocal plot of enzyme reaction with competitive inhibitors
Describe uncompetitive inhibition
Therefore, while Km appears to decrease, indicating a stronger binding affinity between the enzyme and substrate, the enzyme’s capacity to convert substrate to product (Vmax) is still reduced, meaning the overall enzymatic activity is lowered, not increased.
In summary, uncompetitive inhibition reduces overall enzyme activity by decreasing both the apparent Vmax and Km.
What is the significance of uncompetitive inhibition
Describe mixed inhibition (noncompetitive)
Drill rape ts into your mind
What are the two types of cellular enzyme activity controls
Describe covalent and non-covalent modification of enzyme molecules
Describe allosteric control of enzymes
Describe the allosteric control of ATCase
Describe the biosynthesis pathway of CTP
Describe the ATCase holoenzyme structure
Describe the role of ATCase-CTP and the ATCase-PALA in T and R states
How can the catalytic activity of enzymes can be
controlled by covalent modification
How is glycogen phosphorylase controlled
by phosphorylation
Describe phosphorylase as a homodimer
Describe glycogen phosphorylase and AMP
How does the phosphorylation of Ser14 of glycogen
phosphorylase promote T-R transition
Just look at this
