Enzyme Catalysis and Function

### Enzymes
- Enzymes catalyze protein functions.
- Many enzymes' polypeptide chains are sufficient for proper folding.
- Enzymes bind substrate molecules at active sites, forming an enzyme-substrate complex to yield products.
- Coenzymes are organic molecules (like vitamins), and cofactors are metal ions.
  - Apoenzymes lack biological function because they require coenzymes or cofactors that are absent. 
  - Holoenzymes are biologically active enzymes with added coenzymes or cofactors.
- Enzymes accelerate reaction rates without affecting reaction equilibrium.
  - Free energy changes occur from substrate to product molecules, and the difference determines equilibrium.
  - Reaction rate depends on the energy of the transition state (highest energy moment).
- Enzymes lower free energy with the transition state, increasing reaction rates.
- Enzymes reduce free energy through binding and chemical effects.

### Enzyme Binding Effects
- Substrate binding involves interaction with the enzyme and substrate.
  - Water molecules are stripped away, entropy is reduced, and molecules are brought together in a reactive position.
  - Induced fit changes the conformation of the substrate.
  - Substat conformation changes with induced fit
- Transition state stabilization ensures the active site complements the substrate, promoting change into the transition state.
  - Enzymes have high affinities for transition states.
- Transition state analogs bind to the active site with high affinity and act as competitive inhibitors.

### Enzyme Chemical Effects
- Acid/base catalysis involves enzymes picking up or donating protons with substrates.
  - Histidine residues are often involved.
- Covalent catalysis forms a covalent linkage.
  - Involves forming a covalent bond on the substrate molecule in two stages.
  - The second stage is regeneration of the free enzyme
- Serine proteases can demonstrate both acid-base and covalent catalysis.

### Enzyme Kinetics
- Michaelis-Menten plots show velocity versus substrate concentration.
  - Vmax is where the velocity is independent of substrate concentration.
  - 1/2 Vmax equals Km, which measures substrate concentration.
  -  Vo = Vmax[S]/([S] + Km)
  - The visualization is handy but not very accurate because Vmax is often underestimated.
- Lineweaver-Burk plots offer more accurate reciprocal representations.
  - The line vs. the vertical axis represents 1/Vmax.
  - The horizontal axis vs. the line represents -1/Km.

### Reversible Inhibitors
- Competitive inhibitors compete with the substrate for binding to the active site.
  - These resemble the substrate molecule and bind only to free enzymes.
  - Excess substrate washes them out, making competitive inhibitors irrelevant.
  - A higher substrate concentration is needed to reach Vmax, increasing Km.
- Uncompetitive inhibitors bind only to the enzyme-substrate (ES) complex.
  - Substrate binding induces a conformational change, creating an inhibitor binding site.
  - V = [ES]K2, meaning velocity depends on ES complex concentration multiplied by the K2 rate constant.
  - Decreasing the ES complex velocity also decreases velocity overall.
  - More E and S must bind to establish equilibrium.
  - Enzyme affinity for the substrate increases, meaning Km decreases.
- Non-competitive inhibitors can bind to either the free enzyme or the ES complex.
  - The affinity of the enzyme for the substrate is not changed.
  - Vmax decreases because the ES complex concentration decreases.

### Serine Proteases
- Serine proteases cleave polypeptide chains.
  - Trypsin cleaves beside positive residues.
  - Chymotrypsin cuts beside aromatics.
  - Elastase cuts beside small, hydrophobic residues like alanine and glycine.
- Serine proteases Utilize catalytic triad to cut peptides.
  - Chymotrypsin uses acid-base and covalent catalysis.

### Chymotrypsin Stages
- Stage 1: Histidine acts as a base.
  - It extracts a proton from the hydroxyl group of serine.
- Histidine activates the oxygen of the hydroxyl group to attack the carbonyl carbon of the peptide:
  - Histidine acts as an acid, donating a protein to the amide nitrogen to cleave the substrate.
  - The second half still remains linked to the serine
- Stage 2: Histidine acts as a base:
  - It extracts a proton molecule from a water molecule
- The water molecule activates and attacks to a point of covalent linkage on the enzyme and substrate
- Next Histadine is an acid
  - It donates a proton from the water molecule
  - Forms hydroxyl group of the serine

### Regulation of Enzyme Activity
- Activity depends on availability (long term) and direct modification (short term).
- Availability involves controlling the amount of enzyme activity.
  - Enzyme production gets induced and targeted destruction takes place.
- Activity involves covalent modifications, like phosphorylation.

### Covalent Modifications
- Phosphoryl groups are added by kinases.
  - Modifications are reversible.
  - Phosphoryl groups are removed by phosphatases.
- Glycogen regulation involves phosphorylation.
  - When both enzymes are phosphorylated, catalase enzymes are activated.
  - However,  unphosphorylated enzymes lead to anabolic enzyme activation.
- Glucose residues can create a homopolysaccharide called glycogen.
  - Glucose conversion to glycogen involves glucose synthase.
  - When there is insulin, glucose stored as glycogen
- Glycogen conversion to glucose involves glucose phosphorylase,.
  - When hungry/scared, epinephrine or glucagon hormones are present.
  - Both enzymes are phosphorylated.
- Futile cycling occurs when both enzymes are activated at the same time.
  - It is avoided by reciprocal regulation to prevent concurrent catabolic and anabolic enzyme activity.
- Non-covalent regulation (allosteric regulation) takes place

### Allosteric Enzymes
- Allosteric enzymes have multiple binding sites:
  - Active sites that substrates bind to.
  - Allosteric sites where allosteric modulators bind.
- Quaternary structure is large and complex.
  - Two conformations are generally associated with them (R state and the T state).
  - Allosteric modulators influence the equilibrium between the T and R states.
- These are slow and rate-limiting steps between enzymatic pathways.
  - Typically, they catalyze the first unique, committed step and regulate via negative feedback.
- Sigmoidal relationships do not follow Michaelis-Menten kinetics.
  - Similar to hemoglobin's oxygen-binding curve.
  - Sensitivity to substrate concentration changes.
  - Small concentration changes cause large velocity changes, displaying a threshold effect.
- Glycolysis converts glucose into ATP.

### Glycolysis Regulation
- PFK1 (phosphofructokinase-1) is regulated by key allosteric modulators
  - PEP (phosphoenolpyruvate) is an allosteric inhibitor of the pathway's final product.
  - ADP (adenosine diphosphate)is an allosteric activator and indicates a lack of ATP.
- Relative activity depends on PEP and ADP concentrations.
  - Activity increases with ADP > PEP.
  - Activity decreases with PEP > ADP.

### Carbohydrates 
- Hydrates of carbon in molecular formulas mean that for every carbon molecule, one water molecule is associated with it.
  - Chiral carbons are abundant carbohydrates.
- Stereoisomers are determined as 2^n, where n is the number of chiral carbons.
- L or D sugar configuration refers to the chiral carbon farthest from the carbonyl carbon.
  - Epimers describe one chiral carbon that differs. 
  - C4 galactose is an example
- Carbohydrates include ketoses and aldoses.
  - 5-carbon sugar is ribose.
  - 6-carbon sugars are glucose, fructose, galactose.
  - Longer carbohydrates (5+ carbon units) become cyclic structures.
- Hydroxyl groups attach to the carbonyl carbon.
- Non-chiral carbons convert to chiral carbons.
  - The anomeric carbon results from cyclization:
    - At C1 for aldoses
    - At C2 for ketoses
- Anomeric carbon have two different isomers, alpha and beta stereoisomers.
- Alpha and beta versions of anomers of each other
- Mutarotation is the interconversion of alpha and beta forms through a linear intermediate.

### Naming Disaccharides
- Disaccharides always have two 6-carbon aldoses in the pyran ring structure joined together.
- Glucose and galactose are always involved.
- Carbon 4 determines if a disaccharide is glucose or galactose.
  - OH up equals galactose. Conversely, OH down equals glucose
- Reducing end has a free anomeric carbon, also known as free carbon 1.

### Polysaccharides
- Includes homopolysaccharides and heteropolysaccharides.
- Plants store starch for energy.
  - Amylose is unbranched via a(1-4) linkages.
  - Amylopectin is branched, similar to amylose.
    - Branches via a(1-6) linkages at every 24-30 residues.
- Glycogen chains exhibit frequent branching with a(1-4) and a(1-6) linkages.
- There are more non-reducing ends, enabling quicker glucose mobilization.
- Structural polysaccharides use beta linkages instead of alphan linkages.

### Lipids
- Lipids are united by aggregates (non-covalent)
- Fatty acids have a carboxyl group and a hydrocarbon tail:
  - Vary in hydrocarbon tail length and double bond location.
  - Typically 12-24 carbons long, with even numbers of carbons.
  - No double bonds mean saturated fats.
  - One double bond means monounsaturated fats.
  - Multiple double bonds means polyunsaturated fats.
- Fatty acids that form solids are typically long and saturated.
  - Saturated composition is more significant for solidity than the length is.
- Naming fatty acids lists the number of carbons, double bonds, and ∆number of the carbons involved in the double bonds.

### Lipid Energy Storage
- Triacylglycerol links 3 fatty acids to a glycerol backbone through ester linkages.
- Ester linkages occur between the hydroxyl group of glycerol and the carboxyl group of the fatty acid.
- Advantages for energy include low oxidation and hydration states.
  - Large amount of energy per gram.
  - Hydrophobic to not have associated water weight.
- Saponification releases fatty acids from ester linkages through base treatment.

### Membrane Lipids
- Membranes have two hydrocarbon tails + polar head groups.
  - Backbones include glycerol or sphingosine.
    - Sphingosine involves a long-chain amino alcohol with a fatty acid covalently linked through an amide linkage.
- Polar head groups vary and have unique functions.

### Fat-Soluble Vitamins
- D vitamins promote bone formation.
- A vitamins promote vision.
- E Vitamins neutralize free radicals.
- K vitamins promote coagulation.

### Cholesterol
- Cholesterol is a bulky group of planar rings.
- It mediates membrane fluidity.
- It serves as a precursor for many active signaling molecules, including sex hormones and corticosteroids.

### Eicosanoids
- Eicosanoids produce paracrine hormones (act close to the production site).
  - Includes prostaglandins that promote fever and inflammation.
  - Includes thromboxanes that promote blood clot formation.
  - Also includes leukotrienes such as smooth muscle contraction.
- Aspirin blocks prostaglandin and thromboxane, but not leukotriene production.

### Membranes
- Membranes have undergone specialization (different composition of lipids and carbohydrates).
- They are asymmetric.
- They consist of lipids and proteins, with higher protein concentrations signifying a more active membrane.
- Membranes have a fluid mosaic model because membranes are held together by non-covalent forces.
  - They have freedom of movement within the plane of the membrane (lipids and membrane)

### Membrane Protein Variety
- Peripheral proteins associate with the membrane's surface through hydrogen or electrostatic bonds.
- Lipid-linked proteins have hydrocarbon tails covalently linked on them.
  - On the inside of the cell, linked to thyrogen. 
  - On the outside of the cell, are linked to GPI anchors.
- Integral proteins have membrane-spanning regions with hydrophobic residues (24 in length).
  - The sequence of a protein identifies membrane-spanning regions, signifying transmembrane protein.

### Lipid Racks
- Lipid racks are bulges in the membrane of longer hydrocarbon tails
- Sphingolipids tend to be longer hydrogens and stabilize the racks
- They can be spontaneous

### Transport Across Membranes
- Molecules get across the membrane by:
   - Diffusion
         - Simple diffusion occurs for small nonpolar molecules 
   - Facilitated diffusion
        - Channels (a passageway from one side to the other)
        - Carriers (bind and cause the molecule to transport from one side to the other)
   - Active Transport
        - Primary active transport (requires ATP)
             - ATP as energy source
             - P-Type, a phosphorylated intermediate
             - V-Type pumps proteins into vesicles
             - ABC transporters pumps toxins out
        - Secondary active transport (uses molecule gradients as source of energy) 
              - Sodium gradients drive glucose transport into epithelial cells

### Nucleic Acids
- One should be able to identify all the nitrogenous bases.
- Nucleosides differ in their nitrogenous base.
  - Whether or not they have a hydroxyl group.
  - Whether or not they have phosphoryl groups on the 5' carbon of the ribose.
- DNA and RNA strands are formed the same way:
  - Phosphodiester linkages link two different nucleotide molecules from the 5'-3' carbons (each is negatively charged)
  - Nitrogenous bases are linked to the C1 of the ribose.
- DNA and RNA have different stabilities because of the hydroxyl group on the 2'
  - RNA experiences more base hydrolysis.
  - Functions complement each other
- Nucleic acid strands can form higher-order structures.
  - These structures brings together nonpolar nitrogenous bases.

### DNA and RNA Bonding
- Specific use of hydrogen bonding patterns is key.
  - Strands come together in an antiparallel and complementary fashion (known Watson-Crick base pairs)
- Chargaff's rule dictates purine-pyrimidine pairings (AT, GC).
- Duplex DNA is packaged within eukaryotes with histone proteins.
  - Histones are conserved cationic proteins, thus carrying a positive charge
- Nucleosomes core is an 8-polypeptide, H2A, H2B, H3, H4, surrounded by 146 base pairs.
  - H1 binds to the short stretch of DNA.
  - Bond and function are both driven by electrostatic interactions.
- Eukaryotic chromosomes are linear, while bacteria have circular chromosomes
  - Ends are present in eukaryotes
  - Telomeric sequences (repeated sequences) occur because DNA is lost from every replication
  - Introns provide intervening sequences that are spliced out to maintain functional mRNA products.
- Restriction enzymes are part of a prokaryotic defensive system that recognizes/cuts specific sequences in DNA.
  - Common sequences tend to be palindromes.
  - RFLP stands for restriction fragment length polymorphism, for use in forensics.

### Polymerase Chain Reaction
- PCR consists of multiple steps
  -Heating Denatures
  -Cooling Anneals (strands come back together)
  -Heating Stabilizes Polymerase (heat stable polymerase)
- PCR rapidly applies specific regions of DNA.