Enzyme Catalysis and Kinetics

Module 6: Enzyme Catalysis and Kinetics

• Life is dependent on reactions which are catalyzed with speed, specificity, and regulation regarding changing conditions.
• Some enzymes are both sensors and catalytic agents.
• Some enzymes need co-enzymes or co-factors to function catalytically.
• The equilibrium between a substrate and product depends on the difference in free energy between them, with the equilibrium favoring the molecule with lower free energy.
• The rate of interconversion between substrate and product depends on the energy barrier that separates them.
• Enzyme-catalyzed reactions form a complex between the enzyme and its substrate (ES complex).
• Substrate binding happens at the active site, which is located in a pocket on the enzyme.
• Enzymes lower the activation energy for a reaction and thus increase the reaction rate.
• Enzymes do not change the free energy difference between the substrate and product, and thus do not affect the equilibrium of a reaction.
• Circe effect describes how some enzymes catalyze reactions faster than diffusion-controlled rate limits.
• Weak interactions between the substrate and enzyme account for much of the energy utilized for enzymatic rate enhancements.
• Enzyme active sites are structured to favor these weak interactions in the reaction transition state, stabilizing the transition state.
• Covalent and acid-base catalysis are additional catalytic mechanisms used by enzymes.
• Enzymes share certain kinetic properties.
• When a substrate is added to an enzyme, the reaction rapidly achieves a steady state where the rate that the ES complex is formed balances the rate at which it breaks down.
• As the concentration of the substrate [S] increases, activity increases in a hyperbolic fashion towards a maximal rate, Vmax, at which point the enzyme is saturated with the substrate.
• Michaelis-Menton plots relate substrate concentration and velocity, and can be used to determine enzyme Km and Vmax.
• Km is the concentration of the substrate required for an enzyme to function at half the maximum velocity.
• The Michaelis-Menton equation relates to initial velocity to [S] and Vmax using the constant Km.
• The Michaelis-Menton equation derivation is based on the steady-state assumption, where the rate of ES complex formation is equal to the rate of its breakdown.
• Lineweaver-Burke plots are double reciprocal plots (1/V vs 1/[S]) that also consider the relationship between velocity and substrate concentration.
• Lineweaver-Burke plots can be used to determine the Km and Vmax of an enzyme, and are more accurate than Michaelis-Menton plots.
• Enzyme inhibition is categorized into the groups: Competitive, uncompetitive or noncompetitive.
• Competitive inhibitors only bind to the free enzyme (at the active site).
• Competitive inhibitors increase Km, but do not impact Vmax.
• Uncompetitive inhibitors only bind to the ES complex.
• Uncompetitive inhibitors decrease both Vmax and Km.
• Noncompetitive inhibitors bind both the free enzyme and the ES complex.
• Noncompetitive inhibitors decrease Vmax without changing Km.
• Irreversible inhibitors bind permanently to the active site by forming a covalent or stable non-covalent interaction.
• Chymotrypsin is a serine protease with a well-understood mechanism, featuring acid-base catalysis, covalent catalysis and transition-state stabilization.
• Enzymatic pathways are frequently regulated through feedback inhibition, where the end product inhibits the first enzyme of that same pathway.
• The activity of allosteric enzymes is adjusted by the reversible binding of modulators (either activators or inhibitors) to regulatory allosteric sites.
• Allosteric enzymes do not follow Michaelis-Menton kinetics, and instead demonstrate a sigmoidal relationship between velocity and substrate concentration.
• The threshold effect refers to the sensitivity of allosteric enzymes to changes in substrate concentration.
• Other regulatory enzymes are modulated by covalent modification through the reversible addition of a functional group to regulate some function of the enzyme.

Module 7: Sugars

• Sugars are hydrates of carbon, and tend to conform to the formula (CH2O)n
• Sugars are compounds that contain an aldehyde or ketone group and at least two hydroxyl groups.
• Aldoses feature an aldehyde group with the carbon bonded to oxygen positioned at the end of the chain.
• Ketones have a ketose carbon with the carbon double-bonded to oxygen that is not positioned at the end of the chain.
• Monosaccharides have several chiral carbons, and therefore exist in a variety of stereochemical forms.
• The formula 2n yields the number of isomers of a sugar, where n is the number of chiral carbons.
• Epimers are sugars that differ in configuration at a single chiral carbon.
• Sugars are designated as D or L based on the configuration of the chiral carbon most distant from the carbonyl carbon compared to glyceraldehyde.
• Sugars with five carbons or more are usually cyclized.
• Sugars can be cyclized in pyran (six-membered ring) or furan (five membered ring) ring forms.
• Cyclization of a sugar occurs when a hydroxyl group from within the sugar attacks the carbonyl carbon.
• The anomeric carbon is the carbon that becomes chiral as a result of cyclization.
• Anomeric carbons can take the form of alpha or beta isomers.
• Alpha and beta isomers of a cyclized sugar can interconvert through mutarotation.
• Polysaccharides can be hetero or homo, and can be branched or linear.
• Energy storage polysaccharides like glycogen, amylose, amylopectin tend to have alpha 1-4 linkages
• Structural polysaccharides like cellulose and chitin tend to have beta 1-4 linkages.

Module 8: Lipids

• Lipids are water-insoluble molecules of diverse structure and function
• Fatty acids consist of a long hydrocarbon chain with a carboxyl head group.
• Fatty acids usually are made of an even number of carbons (usually 12 to 24), and can be saturated, unsaturated, or polyunsaturated.
• Saturated Fatty Acids – no double bonds in the hydrocarbon tail.
• Unsaturated Fatty Acids – one double bond in hydrocarbon tail.
• Polyunsaturated Fatty Acids – multiple double bonds in hydrocarbon tail.
• Double bonds in the hydrocarbon tails of fatty acids are commonly configured as cis.
• Triacylglycerols contain three fatty acid molecules linked to the three hydroxyl groups of glycerol through ester bonds.
• Simple triacylglycerols only contain one type of fatty acid, while complex triacylglycerols contain two or more types of fatty acids,
• Triacylglycerols are primarily energy storage fats.
• Most membrane lipids have two hydrocarbon tails and a polar head group.
• Glycerophospholipids and the sphingolipids are the most common membrane lipids.
• The most abundant membrane lipids are the glycerophospholipids, which consist of diacylglycerol with a polar head group linked to the third hydroxyl of glycerol through a phosphodiester bond.
• Glycerophosphpolipids differ in the structure of their head group, examples include phosphatidylethanolamine and phosphatidylcholine.
• Sphingolipids are derived from sphingosine, a long-chain aliphatic amino alcohol.
• Ceramides are formed when a fatty acid is linked to the amino group of sphingosine through an amide bond.
• Sphingolipids have a polar head group attached to the ceramide backbone.
• The polar head groups of sphingolipids can be phosphocholine (sphingomyelin) or sugar groups (cerebrosides and gangliosides).
• Sterols feature four fused rings and a hydroxyl group.
• Cholesterol is the major sterol in animals, and is a structural component of membranes as well as a precursor to a wide range of steroids.
• Some lipids play critical roles as cofactors or signals, even when present in relatively small quantities.
• Phosphatidylinositol bisphosphate is hydrolyzed to yield two intracellular messengers: diacylglycerol and 1,4,5-triphosphate.
• Prostaglandins, thromboxanes and leukotrienes (the eicosanoids) are derived from arachidonate, and are potent paracrine hormones.
• Vitamins K, A, D and E are fat-soluble compounds.
• Vitamin D is a precursor to a hormone that regulates calcium metabolism.
• Vitamin A furnishes the visual pigment within the eye.
• Vitamin E functions in the protection of membranes from oxidative damage.
• Vitamin K is essential in the blood-clotting process.

Module 9: Biological Membranes

• Biological membranes define cellular boundaries, divide cells into separate compartments, organize complex reaction sequences, and act in signal reception and energy transformations.
• Membranes are composed of lipids and proteins in varying combinations, which varies depending on species, cell types, and organelles.
• The fluid mosaic model describes the ability for membrane lipids and proteins to move laterally within the membrane.
• The lipid bilayer is the basic structural unit of membranes.
• Fatty acyl chains of membrane lipids are oriented toward the interior of the bilayer, and their hydrophobic interactions stabilize the bilayer but give it flexibility.
• Peripheral membrane proteins are loosely associated with the membrane through electrostatic interactions and hydrogen bonds.
• Integral membrane proteins are strongly associated with membranes, and span through the membrane via hydrophobic interactions between the lipid bilayer and their non-polar amino acid side chains.
• Lipid-linked proteins have hydrocarbon chains that are covalently linked, which anchor the protein to the membrane face.
• Membrane spanning regions of integral membrane proteins often consist of a stretch of 20 hydrophobic amino acids, which can be predicted using the primary structure of the protein.
• The lipids and proteins of membranes are inserted into the bilayer with specific sidedness, which gives the membranes structural and functional asymmetry.
• Membrane fluidity changes depending on temperature, fatty acid composition, and sterol content.
• Membranes can exist in liquid-ordered or liquid-disordered states.
• In liquid-disordered states, thermal motion of acyl chains makes the interior of the bilayer fluid.
• Flip-flop diffusion of lipids between the inner and outer face of membranes is very slow unless catalyzed by flippases.
• Lipids and proteins can diffuse laterally within the plane of the membrane, but this mobility can be limited by interactions of membrane proteins with internal cytoskeletal structures and interaction of lipids with lipid rafts.
• Movement of polar compounds and ions across biological membranes requires protein transporters.
• Some transporters simply facilitate passive diffusion across the membrane from the side of higher concentration to the side of lower concentration.
• Other transporters bring about active movement of solutes against a gradient, and requires energy.
• Primary active transport uses the energy of ATP to move molecules against a concentration gradient.
• Secondary active transport, involves coupled flow of two solutes, where one flows down its electrochemical gradient as the other is pulled up its gradient.
• In animal cells, Na K ATPase maintains the differences in cytosolic and extracellular concentrations of N and K.
• The resulting Na gradient for Na K ATPase is used as the energy source for a variety of secondary active transport systems.
• Ion channels enable rapid movement of ions across the membrane.
• Ion channels can be voltage or ligand-gated.
• Ion channels differ from ion transporters in their greater speed, absence of saturation limits, and regulation.

Module 10: Nucleic Acids and Energy

• A nucleotide consists of a nitrogenous base (purine or pyrimidine), a pentose sugar, and at least one or more phosphate groups.
• Nucleic acids are polymers of nucleotides, joined together by phosphodiester linkages between the 5’-hydroxyl group of one pentose and the 3'-hydroxyl of the next pentose.
• RNA and DNA are the two types of nucleic acid.
• The nucleotides in RNA contain ribose.
• The nucleotides in DNA contain 2'-deoxyribose.
• Watson and Crick stated that native DNA consists of two anti-parallel chains in a right-handed double-helical arrangement.
• Within the double helical DNA, complementary base pairs A with T and G with C are formed by hydrogen bonding.
• The base pairs of DNA are stacked perpendicular to the long axis of the double helix, 3.4 Å apart, with 10.5 base pairs per turn.
• Messenger RNA encodes genetic information from DNA to ribosomes for protein synthesis.
• Transfer RNA and ribosomal RNA are also involved in protein synthesis.
• RNA can be structurally complex.
• Single RNA strands can be folded into hairpins, double-stranded regions or complex loops.
• Native DNA undergoes reversible separation of strands (melting) on heating.
• DNAs rich in GC base pairs have higher melting temperatures than DNAs rich in AT base pairs.
• ATP is the central carrier of chemical energy in cells.
• The presence of an adenosine moiety in a variety of enzyme cofactors may be related to binding-energy requirements.
• Cyclic AMP, formed from ATP in a reaction catalyzed by adenylyl cyclase, is a common second messenger produced in response to hormones.
• Restriction enzymes cut double stranded nucleic acid molecules at specific sequences, which are typically palindromes or self-complimentary about a point.
• Restriction enzymes are a natural bacterial defense system and are utilized in manipulations of DNA.
• The polymerase chain reaction (PCR) can be used to amplify selected DNA segments from a DNA library or en entire genome.
• Genes are segments of a chromosome that contain the encoding information for a functional polypeptide or RNA molecule.
• Chromosomes also contain a range of regulatory sequences which are involved in replication, transcription and other processes.
• Genomic DNA and RNA molecules are usually longer than the viral particles or cells that contain them.
• Many genes in eukaryotic cells are interrupted by noncoding sequences called introns.
• The coding segments that are separated by introns are called exons.
• Eukaryotic chromosomes have two important function repetitive DNA sequences: centromeres and introns
• Centromeres – attachment points for mitotic spindles.
• Introns – located at the ends of chromosomes.
• Histones are highly conserved, basic proteins involved in DNA packaging in eukaryotes.
• The fundamental unit of organization in the chromatin of eukaryotic cells is the nucleosome core particle which consists of histones and a 146 bp segment of DNA wrapped around eight histone proteins (two copies each of histones H2A, H2B, H3 and H4).
• Histone H1 is a linker histone that binds to the DNA between nucleosomes.