Enzymes and Binding Effects

Enzymes

• Enzymes catalyze protein
• Many enzymes can fold properly from just their polypeptide form
• Enzymes form enzyme-substrate complexes by binding substrate molecules at active sites
• Coenzymes are organic molecules like vitamins and cofactors are metal ions
  • Apoenzymes lack biological function and require coenzymes or cofactors to function
  • Holoenzymes are biologically active enzymes with coenzymes or cofactors added
• Enzymes accelerate reaction rates without affecting equilibrium
  • Equilibrium is determined by the difference in free energy between substrate and product molecules
  • Reaction rate is determined by the energy of the transition state
• Enzymes lower free energy by stabilizing the transition state and increasing reaction rates
• Enzymes lower free energy through binding and chemical effects

Binding Effects

• Substrate binding involves interaction with the enzyme, stripping water, and reducing entropy
• Binding brings molecules together and induces fit by changing the substrate's conformation
• Transition state stabilization
  • Active sites are complimentary to substrates, allowing for specificity
  • Active sites promote changes into the transition state
  • Enzymes have high affinities for transition states
• Transition state analogs bind with high affinity and inhibit enzymes

Chemical Effects

• Acid/base catalysis involves enzymes donating or accepting protons, often with histidine
• Covalent catalysis involves forming a covalent linkage in two stages
  • Covalent bond formation on the substrate
  • Regeneration of the free enzyme, releasing the attached part
• Serine proteases use both acid/base and covalent catalysis

Enzyme Kinetics

• In Michaelis-Menten plots, Vmax denotes the point where velocity is independent of substrate concentration
  • Km measures substrate concentration at 1/2 Vmax
  • Vo=Vmax[S]/([S]+Km)
• Lineweaver-Burk plots are double reciprocal representations
  • Vertical axis represents 1/Vmax
  • Horizontal axis represents -1/Km

Reversible Inhibitors

• Competitive inhibitors
  • Bind to the active site
  • Resemble the substrate molecule
  • Only bind to free enzyme
  • Are irrelevant in excess substrate
  • Increasing substrate raises Vmax, so Km increases
• Uncompetitive inhibitors
  • Bind only to the enzyme-substrate (ES) complex
  • Binding causes a change and creates an inhibitor binding site
  • Velocity depends on ES complex concentration and K2 rate constant
  • Decreasing ES complex velocity causes more E and S to bind to make more ES complex for equilibrium
  • Enzyme affinity for the substrate can seem to rise as Km decreases
• Non-competitive inhibitors
  • Bind to free enzyme or the ES complex
  • Do not change the affinity of the enzyme for the substrate
  • Decreasing concentrations of ES complex decreases the Vmax

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 use a catalytic triad to cut peptides, using both acid-base and covalent catalysis

Chymotrypsin Stages

• Stage 1
  • Histidine extracts a proton from serine to activate the oxygen on the hydroxyl group, allowing for attack on the carbonyl carbon of the peptide
  • Histidine donates its proton to the amide nitrogen, cutting the substrate in two
• Stage 2
  • Histidine extracts a proton from a water molecule, activating the water's oxygen to attack the enzyme-substrate covalent linkage
  • Histidine donates its proton to regenerate the serine hydroxyl group

Enzyme Activity Regulation

• Availability (long term): controlling the amount of enzyme activity via induced production or targeted destruction
• Activity (short term): covalent regulation through phosphorylation via kinases, which is reversible via phosphatases
• Glycogen regulation
  • Phosphorylated: catalase enzyme is active
  • Unphosphorylated: anabolic enzyme is active
• Glycogen creation and breakdown
  • Glucose to glycogen is anabolic with glucose synthase. Insulin presence leads to glucose storage as glycogen
  • Glycogen to glucose is catabolic with glucose phosphorylase. Signaled by epinephrine or glucagon when hungry/scared
• Futile cycling is avoided to prevent simultaneous activation by reciprocal regulation
• Non-covalent regulation occurs through allosteric regulation

Allosteric Enzymes

• Allosteric enzymes have multiple binding sites for substrates and allosteric modulators
• Quaternary structure is large and complex and tends to have two conformations (R and T states)
• Allosteric modulators influence equilibrium between T and R states and tend to be slow
• Allosteric enzymes are rate-limiting steps that catalyze the first unique and committed steps and regulate via negative feedback

Kinetics and Glycolysis

• Allosteric enzymes have sigmoidal relationships and do not obey Michaelis-Menten kinetics
• Hemoglobin's oxygen-binding curve is similar
  • Small substrate concentrations can cause large changes in the allosteric enzyme's velocity, creating a threshold effect
• Glycolysis converts glucose to ATP
  • PFK1 is regulated by PEP as an allosteric inhibitor and ADP as an allosteric activator
  • Relative activity depends on PEP and ADP concentrations
    • Activity is fastest when ADP is greater than PEP and slowest when PEP is greater than ADP

Carbohydrates

• Carbohydrates are hydrates of carbon with the molecular formula (CH2O)n
• Carbohydrates have many chiral carbons
  • Stereoisomers are determined by 2^n where n is the number of chiral carbons
• L or D configuration is determined by the chiral carbon furthest from the carbonyl carbon
• Epimers differ at one chiral carbon
• Carbohydrates can be ketoses or aldoses

Sugars

• Ribose five-carbon sugar
• Glucose, fructose and galactose are six-carbon sugars
• Carbohydrates that contain 5+ carbons will form cyclic structures
  • Carbon becomes chiral
  • Cyclization leads to anomeric carbon
    • Anomeric carbon is C1 in aldoses
    • Anomeric carbon is C2 in ketoses
  • Anomeric carbon creates α and β stereoisomers

More Complex Sugars

• Mutarotation involves interconversion of alpha and beta isomers
• Naming of disaccharides involves two six-carbon aldoses together in a pyran ring linked together
  • Will always be glucose or galactose as building blocks
  • Look at the carbon 4 arrangement
    • OH up: Indicates Galactose
    • OH down: Indicates Glucose
  • Can have reducing ends with a free anomeric carbon
• Polysaccharides include homopolysaccharides and heteropolysaccharides
  • Storage for energy amylose amylopectin
    • Plants store starch
      • Amylose is glucose linked by α(1-4) linkages
      • Amylopectin has α(1-6) branches roughly every 24-30 residues
  • Glycogen has α(1-4) and α(1-6) branch linkages with a greater frequency of branch points
    • More branches allow faster mobilization by cleaving more glucose
  • Some structural polysaccharides incorporate β-linkages, instead of α-linkages

Lipids

• Lipids form aggregates united by noncovalent forces
• Fatty acids consist of a carboxyl group with a hydrocarbon tail, which varies in length and number of double bonds
  • Tail length is 12-24 carbons (usually even)
    • No double bond: saturated
    • 1 double bond: unsaturated
    • Multiple double bonds: polyunsaturated
• Saturated fatty acids of longer length are more likely to solidify

Lipids Cont.

• Naming fatty acids involves the number of carbons, number of double bonds, and position of the carbons involved in double bonds
• Lipids store energy
  • Triacylglycerols link three fatty acids to a glycerol backbone and ester linkage, which forms between the hydroxyl of the glycerol and carboxyl of the fatty acid
    • Advances in terms of energy storage and low hydration state
    • Store more energy per gram
    • They are hydrophobic and carry no water weight

Lipid Function

• Process to release treatment with a base to separate fatty acids from a treated ester
• Membrane lipids have polar head groups attached to 2 hydrocarbon tails
  • Have different backbones
    • May be glycerol or sphingosine (has a long amino alcohol covalently linked to a fatty acid by an amide bond)
  • Have different polar head groups, giving specialized function
• Fat-soluble vitamins
  • Vitamin D builds bone
  • Vitamin A maintains vision
  • Vitamin E neutralizes free radicals
  • Vitamin K causes coagulation
• Cholesterol is a bulky planar ring group that mediates membrane fluidity and serves as a precursor for signaling molecules like sex hormones and corticosteroids

Eicosanoids and Membranes

• Eicosanoids act as paracrine hormones produced near their site of action
  • 3 eicosanoids
    • Prostaglandins signal fever and inflammation
    • Thromboxanes enable blood clot formation
    • Leukotrienes contract of smooth muscle
  • Aspirin inhibits prostaglandin and thromboxane production
• Membranes
  • Undergo specialization with different carbohydrate and lipid compositions
  • Asymmetric structure

Membrane Structure

• Membranes form from lipids and proteins with concentrations dependent on functionality - Higher concentration in membrane for more active proteins
• Fluid mosaic model describes a membrane of non-covalent forces allowing movement of components
• Different membrane protein types
  • Peripheral proteins associate with membrane surfaces through hydrogen bonds or electrostatically
  • Lipid-linked proteins have hydrocarbon tails covalently linked. One type is linked to a thyrogen inside, another is linked to GPI anchors outside
  • Integral proteins contain hydrophobic residues in membrane-spanning regions of about 24 residues in length
    • Transmembrane protein sequences can predict spanning regions
• Lipid racks
  • Bulges in the membrane structure where lengthier hydrocarbon tails cluster
  • Sphingolipids make the structure sturdy
  • Are spontaneous

Transport Across Membranes

• Molecules cross membranes through:
  • Diffusion: Small, nonpolar molecules directly pass
  • Facilitated diffusion: Utilizes channels (passageways) and carriers (bind and transport molecules)
  • Active transport:
    • Primary: requires ATP. P-type uses phosphorylated intermediate, V-type pumps proteins in vesicles, and ABC transporters pump out toxins
    • Secondary: uses a gradient provided when taking glucose into epithelial cells using a sodium gradient

Nucleic Acids

• Nucleo bases differ in groups attached
• Nucleosides groups differ in whether or not they also have attached 5' ribose hydroxyl or phosphoryl groups
• DNA and RNA strands constructed the same way
  • Phosphodiester is a 5'-3' linkage with two negative charges between two nucleotides
  • Carbon 1 of ribose link to a nitrogenous base

Nucleic Acid Properties

• DNA and RNA differ in stability due to the hydroxyl group on the 2’ carbon (RNA is more susceptible to base hydrolysis)
• Nucleic acids can form higher-order structures using nonpolar nitrogenous bases
• Specific structures depend on the hydrogen-bonding patterns of the nitrogenous bases
  • Strands are antiparallel and complementary
  • Base pairs depend on having one thing happen when the other is occurring
• Chargaff's rule: purines pair with pyrimidines (A-T, G-C)

DNA Compaction

• Compacting duplex DNA is done with histone proteins

  • Help to make duplex DNA in eukaryotes

• Histones are highly-conserved cationic proteins with positive charge

• Nucleosome cores consist of eight polypeptides (H2A, H2B, H3, H4) wrapped around 146 base pairs, with histone H1 binding to the short DNA stretch in between

• Core interactions are driven by electrostatic forces

• Chromosomes - Are linear, while bacteria are circular - Have endpoints and telomeric repeats - Can have introns which intervening sequences that need to be spliced out to produce the functional mRNA molecules

• Restriction enzymes exist in the context of cutting specific sequences in DNA

DNA Methods

• Can recognize sites in a prokaryotic defensive system

  • Tend to cut out palindrome sequences
  • RFLP in forensics

• PCR amplifies specific regions of DNA

  • A denature to heat up
  • Reanneal on cooldown
  • Elongation is carried out with heat-stable polymerase