Enzymes: Function and Mechanism

Enzymes

• Function as protein catalysts.
• Polypeptide folding is adequate for several enzymes to operate properly.
• Combining with substrate molecules at the active site results in an enzyme-substrate complex, which then yields products.
• Coenzymes (organic molecules like vitamins) and cofactors (metal ions) are important.
• Enzymes lacking necessary coenzymes/cofactors are termed apoenzymes and lack biological function.
• Holoenzymes are created when coenzymes or cofactors are introduced, rendering enzymes biologically active.
• Though accelerating reaction rates, Enzymes do not affect reaction equilibrium.
• The free energy differential between substrate and product indicates equilibrium.
• The transition state's energy dictates the reaction rate.
• Enzymes accelerate reactions by decreasing free energy at the transition state.

Enzyme Function

• Enzymes reduce free energy through chemical and binding effects.

Binding Effects

• Substrate binding involves interaction between the enzyme and substrate, stripping water, lowering entropy, and uniting molecules in the reactive position.
• Induced fit changes the substrate's conformation.
• Transition state stabilization occurs when the active site complements the substrate, triggering a change in the substrate to encourage transition.
• Compared to substrates, enzymes show greater affinity for transition states.
• Transition state analogs attach to the enzyme's active site with high affinity, serving as competitive inhibitors.

Chemical Effects

• Acid/base catalysis involves the enzyme picking up or donating protons, generally involving histidine residues.
• Covalent catalysis involves a covalent linkage.
• Creation of a covalent bond on the substrate molecule, breaking it into two stages: the covalent bond's formation and free enzyme regeneration.
• Both reactions are observed in serine proteases.

Enzyme Kinetics

• The Michaelis-Menten plot graphs velocity against substrate concentration and Vmax is the point at which velocity is independent of substrate concentration.
• Km measures substrate concentration where the velocity is half of Vmax.
• The equation for velocity is V0 = Vmax[S]/([S] + Km).
• Lineweaver-Burk plots are more accurate for visualization using double reciprocals.
• The vertical axis represents 1/Vmax while the horizontal axis is -1/Km.

Reversible Inhibitors

• Competitive inhibitors compete with the substrate for the active site, resembling the substrate and only binding to free enzyme.
• Competitive inhibitors are irrelevant when washed out by excess substrate and Km increases, requiring more substrate to reach Vmax.
• Uncompetitive inhibitors exclusively attach to the ES complex.
• Substrate binding causes a conformational change that induces an inhibitor binding site.
• Velocity is calculated as V = [ES]K2.
• Decreasing the ES complex velocity also decreases velocity.
• More E and S are required to bind to establish equilibrium, increasing the enzyme's affinity and decreasing Km.
• Non-competitive inhibitors can attach to free enzyme or the enzyme-substrate complex, not changing the substrate's affinity.
• Vmax is decreased by lower ES complex concentration.

Serine Proteases

• Serine proteases cleave polypeptide chains with Trypsin cleaving beside positive residues.
• Chymotrypsin cuts beside aromatics.
• Elastase cleaves next to small hydrophobic residues like alanine and glycine, using a catalytic triad.
• Acid-base and covalent catalysis take place during chymotrypsin's first stage.
• Histidine acts as a base, taking a proton from serine's hydroxyl group and facilitates the oxygen of the hydroxyl group's attach on the carbonyl carbon of the peptide.
• Histidine then becomes an acid, passing on the protein to amide nitrogen to cut the substrate into two parts.
• In stage 2, histidine functions as a base, extracting a proton molecule from a water molecule to activate the water's oxygen.
• Histidine then donates a proton to restore the hydroxyl group of serine.

Regulation of Enzyme Activity

• Availability is long term and controls the amount of enzyme activity by inducing production or targeting destruction of enzymes.
• Activity is short term and uses covalent phosphorylation.
• Kinases add phosphoryl groups.
• Phosphatases remove phosphoryl groups, which allows for reversible modifications.
• Glycogen regulation: Phosphorylation activates catalase enzymes while unphosphorylated enzymes are anabolic.
• Glucose residues can form glycogen through glucose synthase which is anabolic.
• In the presence of insulin, glucose is stored as glycogen.
• Glucose phosphorylase (catabolic) converts glycogen into glucose.
• Epinephrine or glucagon indicates hunger or threat, phosphorylating both enzymes.
• Futile cycling occurs when both enzymes activate at the same time.
• Reciprocal regulation prevents futile cycling to not engage both catabolic and anabolic enzymes simultaneously.
• Non-covalent allosteric regulation exists.
• Allosteric enzymes have various binding sites, including active sites and allosteric modulator binding sites.
• Possessing quaternary structure, these enzymes exhibit two conformations: R state and T state, and operate slowly.
• During the enzymatic pathway, they influence the equilibrium between the R and T states and serve as rate-limiting steps.
• They typically catalyze the first unique and committed step, controlled by negative feedback from the final product.
• These enzymes demonstrate a sigmoidal relationship rather than adhering to Michaelis-Menten kinetics and function is affected by the concatenation of the substrate in small concentrations.
• During glycolysis, glucose is converted to ATP with PEP acting as an allosteric inhibitor and ADP functions as an allosteric activator.
• Activity is related to the relative concentrations of PEP and ADP.

Carbohydrates

• Carbohydrates are hydrates of carbon, following a molecular formula of one water molecule per carbon.
• Chiral carbons are abundant.
• Stereoisomers = 2^n, where n = the number of chiral carbons.
• L or D sugar is determined by the chiral carbon furthest from the carbonyl carbon.
• Epimers differ by one chiral carbon.
• Carbohydrates can be ketoses or aldoses with 5 carbon sugars like ribose and 6 carbon sugars like glucose, fructose, galactose.
• Longer carbohydrates (5+ carbons) tend to form cyclic structures where hydroxyl groups attach to the carbonyl carbon.
• Carbon becomes chiral through cyclization, which produces anomeric carbons.
• C1 for aldoses and C2 for ketoses.
• Alpha and beta stereoisomers form at the anomeric carbon.
• Anomers are alpha and beta versions of each other.
• Mutarotation involves interconversion of the alpha and beta forms via a linear intermediate.
• Naming disaccharides involves 2-6 carbon aldoses in the pyran ring structure.
• It will always be either glucose or galactose.
• Check carbon 4 is either glucose or galactose to determine their identity.
• The reducing end possesses a free anomeric carbon.
• Polysaccharides can be homopolysaccharides or heteropolysaccharides.
• In plants, amylose (unbranched) and amylopectin (branched) are starches.
• Amylose contains glucose residues linked through a(1-4) linkages.
• Amylopectin branches every 24-30 residues and Glycogen has more branch points than amylopectin.
• Glucose stores can be quickly mobilized because non reducing ends are where glucose is cleaved off.
• Structural polysaccharides utilize beta linkages instead of alpha.

Lipids

• Lipids form structures linked by noncovalent aggregation.
• Fatty acids consist of a carboxyl group attached to a hydrocarbon chain whose length and double bond placement vary.
• They typically range from 12-24 carbons and have an even number of carbons.
• Saturated fatty acids have no double bonds.
• Unsaturated fatty acids have one double bond.
• Polyunsaturated have several double bonds.
• Saturated fatty acids with longer chains are solids.
• Naming fatty acids = # of carbons: # of double bonds: ∆number of carbons in the double bonds.
• Lipids serve as energy storage via triacylglycerol, linking three fatty acids to a glycerol backbone through ester linkages.
• High levels of energy storage is achieved with a low hydration and oxidation state.
• They provide more energy per gram and are more hydrophobic.
• Saponification releases fatty acids from ester linkages using base treatment.
• Membrane lipids feature two hydrocarbon tails and polar head groups.
• Backbones differ in that they have either glycerol or sphingosine.
• Polar head groups differ, allowing for specialized functions.
• Fat soluble vitamins include D (bone formation), A (vision), E (neutralizing free radicals) and K (coagulation).
• Cholesterol is a bulky group of planar rings that mediates membrane fluidity and serves as a precursor for active signalling molecules like sex hormones and corticosteroids.
• Eicosanoids are paracrine hormones which includes prostaglandins (fever and inflammation), thromboxanes (blood clots), and leukotriene (smooth muscle contraction).
• Aspirin inhibits prostaglandin and thromboxane production.

Membranes

• Membranes undergo specialization with different composition of lipids and carbohydrates.
• Membranes have asymmetry.
• Membranes are made of lipids and proteins in concentrations varying based on function.
• Fluid mosaic model = membranes are held together by non covalent forces, and move freely within the membrane.
• Peripheral proteins associate with either face of the membrane connected through hydrogen bonds or electrostatically.
• Lipid-linked proteins have hydrocarbon tails covalently linked onto them within the cell via a single thyrogen and outside the cell via GPI anchors in lipid racks.
• Integral proteins span the membrane with 24 hydrophobic residue regions within membrane spanning regions.
• Lipid racks show as bulges where there are membrane lipids of longer hydrocarbon tails.
• Sphingolipids stabilize the racks by having longer hydrogens.
• Molecules cross the membrane by diffusion through small, nonpolar molecules.
• Facilitated diffusion uses channels and carriers.
• Channels are passageways and carriers bind to molecules to transport them to another side.
• Active transport uses energy where the source of energy is ATP.
  • P-Type: phosphorylated intermediate.
  • V-type: pump proteins into vesicles.
  • ABC transporters pump toxins out of cells.
• Secondary active transport uses the gradient of molecules as a source of energy and takes glucose into epithelial cells.

Nucleic Acids

• Identify the the nitrogenous bases.
• Nucleosides differ in their nitrogenous base, whether or not they have a hydroxyl group, or phosphoryl groups on the 5’ carbon of the ribose.
• DNA and RNA strands form the same way through phosphodiester linkages from the 5’-3’ carbons of two different nucleotide molecules.
• The nitrogenous bases are linked to the C1 of the ribose.
• DNA and RNA stabilities vary due to the hydroxyl group on the 2' carbon.
• RNA is more susceptible to base hydrolysis.
• Compliment their functions.
• Nucleic acid strands form higher-order structures using nonpolar nitrogenous bases and are specific through hydrogen bonding patterns.
• Strands come together are antiparallel and complementary.
• Chargaff's rule = purine pairs with a pyrimidine (AT, GC).
• Duplex DNA requires packaging with histone proteins and histones are cationic proteins with a positive charge.
• Nucleosomes cores are made of 8 polypeptide (H2A, H2B, H3, H4) wrapped around 146 base pairs.
• Histone H1 binds to the short stretch of DNA.
• Their interaction is driven by electrostatic interactions.
• Bacteria have circular chromosomes that are linear.
• Chromosomes have endpoints - Telomeric sequences (repeated sequences) because DNA is lost.
• Eukaryotes have introns that are spliced out to produce mRNA molecules.
• Restriction enzymes exist in the context of a prokaryotic defensive system and recognize a specific sequence, and cut the DNA.
• Sequences tend to be palindromic and RFLP is used with forensics.

PCR

• Heating up = denature.
• Cool = anneal (strands come back together).
• Heat = heat stable polymerase.
• Rapidly apply specific regions of DNA.