Biochem 4511 Exam 2 Study
3 Assumptions of Michaelis-Menten
1. ES formation follows rules for equilibria. 2. Ignore reversion of P to ES. 3. Steady state assumption. [ES] is constant as long as [S]>[E]
Enzymes (overview)
1. Higher rates 10^8-12 faster 2. Milder rxn conditions: normal pressure and neutral pH 3. Greater rxn specificity 4. activity can be regulated by substrates, products, etc.
Membrane Lipids
1. Phospholipids 2. Glycolipids 3. Cholesterol
Classes of Enzyme Rate Enhancement
1. Proximity, orientation, and entropy reduction 2. Transition state stabilization 3. Acid-base catalysis 4. Covalent catalysis 5. Metal ion catalysis
Cholesterol in membrane
1. Rigid structure reduces movement of fatty acid tails and forces extended conformation 2. large planar structure prevents close packing 3. broadens temp range by supporting ordered liquid state
Forms of Competitive Inhibition
1. Substrate analogs 2. Transition State analogs (Tamiflu, relenza, Rapivab) 3. Product Inhibition (Telaprevir)
3 functions of Metal Ions
1. binding to substrates to orient for rxn 2. Redox reaction by changing metal oxidation state 3. Electrostatic stabilization or shielding of negative charges Can make bound water molecules acidic (source of OH-)
Hemoglobin
2A and 2B subunits with 4 different O2 binding sites
Phase Transitions
A temperature (transition temperature). Above, the membrane is liquid or fluid. Below, it is (paracrystalline) gel-like solid (thicker).
Microfilaments
Actin microfilaments
Lock and Key
Active site is perfect match for substrate. Modest entropy reduction
Elastase
Ala, Gly, and Val, but mostly Val
Phospholipid
Alcohol head group 4 components: fatty acid tail group, platform that fatty acid is attached to (glycerol), phosphate group, alcohol attached to phosphate Know phosphatidyl serine, choline, and
Electronic complementarity
Also must fit hydrophobicity, charge, H-bonding
Serine protease
Amide bond is very difficult to break. Uncatalyzed-boil overnight in 6M HCl. Catalyzed-protease activity at physiological pH and temperature can break down.
Chymotrypsin
Aromatic hydrophobic residues
Scurvy
Ascorbic acid needed for Hyp conversion. Vitamin C is actually needed to reduce Fe3+ back to Fe2+ (enzyme reactivation) after Hyp is formed.
Catalytic Triad
Asp102-His57-Ser195. Strong H-bond between Asp and His so a proton is shared equally. His acts as base and removes proton from Ser hydroxyl group. After deprotonation, Asp stabilizes positive charged His. Structurally conserved. Convergent evolution
Penicillin Resistance
B-lactamase can inactive penicillin before reaching target enzyme
Trypsin
Basic residues: Lys, Arg
Suicidal Inhibitor
Begin to react but stuck in active site
Transition State Stabilization
Bind to ES with more affinity than to substrates or products.
Allostery (cooperative binding)
Binding of 1st ligand induces conformational change (increased stability) in 2nd unit. 2nd ligand then binds with higher affinity. Allostery can decrease or increase affinity
Uncompetitive Inhibition
Binds directly to ES complex affecting catalytic function. Both Km and Vmax are affected. Lineweaver: parallel lines Additional substrate cannot outcompete because substrate is already bound to ES.
Mixed and Noncompetitive Inhibition
Can bind to enzyme or ES. Noncompetitive is when Km doesn't change (equilibrium between EI and ESI).
Keratin (cont)
Can stretch by pulling and unwinding alpha-helix. With more force, ppt bonds can snap or break.
Carbon Dioxide (increase O2 release directly)
Carbamates form salt bridges stabilizing T-state. In high CO2, transition to T and release of O2. CO2 high in tissues where O2 needed
Fatty Acids
Carboxylic acid with long hydrocarbon tail. Even # of C usually C16 or C18. Double bonds are usually cis and not conjugated (separated by at least a methylene group).
Substrate Preferences
Chymotrypsin, trypsin, and elastase have differing binding pockets P1/S1 pairing.
Phospholipase
Cleave phospholipids at specific sites
Keratin
Coiled-coils making up oligomers! Sulfur content defines rigidity, hydrophobic 1st and 4th residues. Intermediate filament (solely structural)
Competitive Inhibition
Competes with substrate for binding to active site. Vmax doesn't change because high [S] will eventually out compete inhibitor. Km is shifted to aKm (bigger #=weaker binding) Lineweaver: same y, different slope and x-intercept
Schiff base
Conversion of carbonyl to schiff base (imine) allows protonated N to act as electron sink, thus lowering TS energy and increasing rate. Ex. Acetoacetate to acetone. Schiff base avoids unfavorable high energy Enolate state.
Lock & Key
Dihydrofolate reductase with NDAP+ and tetrahydrofolate
Membranes are asymmetric
Distribution is different between outer and inner monolayer
ABC Continued
Enzyme can serve as both acid or base in a single cycle ensuring return to original state. Ex. RNA hydrolysis with 2 His residues each as acid or base
Stickase
Enzyme complementary to transition state is best
Waxes
Esterified Fatty Acids Long chain fatty acids (C14-C36) esterified with long chain alcohol High melting points and extremely hydrophobic
Bilayer forming ability
Fatty acids have single tailed wedge shape. Glycerophospholipids have double tailed and can. Triacylglycerols have 3 tails and can't.
Metal Ion Catalysis
Fe, Cu, Mn, Mo, Co Structural Na, K, Ca Structural and Functional Zn, Mg
Oxyanion Hole
Filled by tetrahedral intermediate. Amide protons form 2 more H-bonds. A 3rd H-bond from N of His.
Sickle Cell Anemia
Glu6 to Val6 in B subunit (EV6); Hemoglobin S (less soluble when deoxygenated so it aggregates); sickling into filaments
Collagen (triple helix)
Gly-Pro-Hyp. Left-handed coils that coil together forming a right-handed coil. High tensile strength and resistant to compression. Crosslinks increase with age
Dissociation Constant
Half saturation; higher Kd=weaker ligand interaction & vise versa
Induced Fit
Hexokinase and glucose
Deacylation
Histidine acts a general base to deprotonate water into OH that reforms tetrahedral intermediate stabilized by oxyanion hole. His then acts as general acid to collapse tetrahedral intermediate.
Sphingomyelin
Hydroxyl group esterified to phosphorylcholine
Preferential Transition State Binding
Increased affinity=increased rate of reaction. 2 more H-bonds=10^6 enhancement
Transition Temperature
Increases with chain and degree of saturation. More saturated lipids needed to maintain membrane integrity at high temperatures.
Enzyme Inhibition
Irreversible, targeted inhibition: suicidal inhibitor
Penicillin
Irrversibile Inhibitor inhibits transpeptidase that forms a peptide bond between 2 peptides (2 alanines) Penicillin resembles transition state
Dissociation Constant (Kd)
Kd=[P][L]/[PL]
Noncompetitive
Lineweaver: Vmax is reduced by a-factor. Same x intercept but different slope and y intercept.
Mixed
Linweaver: Km and Vmax changes. Different x and y and slope. Crosses at some point but not on an axis.
Proximity, orientation, and entropy reduction
Local concentration and proper alignment. 1. Brings substrates into contact with catalytic groups (<5 fold increase) 2. Enzymes bind substrates in proper orientations (100-fold increase) 3. Enzyme freezes substrate rotational and translational motions (holds substrate still). Up to 10^7. Large entropic penalty offset by binding energy of ES complex. ES complex reduces G.
Importance of mixed lipid composition
Maintain membrane fluidity over range of temperature Ex. low temperature adaptation or lipid rafts
ES Complex
Many weak interactions between enzyme and substrate that offset the cost of an unfavorable transition state. Steric strain favors transition state.
Lipid rafts
Membrane subdomains high in cholesterol and glycosphingolipids which have large head groups. Float and diffuse as a group
Transition State Analogs (Inhibitors)
Molecule that looks like transition state. Ex. proline Isomerase has 2 analogs that inhibit catalysis of L to D-proline.
Specificity
Most enzymes can act on range of similar substrates. Ex. Alcohol dehydrogenase acts on all alcohols but best with ethanol
Non-Michaelis Reactions
Multisubstrate enzymes like transferases Ex. Transketolase: ping-pong mechanism. Non hyperbolic curve/cooperative behavior of oligomeric enzyme
Molecular Evolution
Myoglobin first, then alpha and beta, then the rest
Zymogens
Nonspecific proteases carefully inhibited. Intestinal protease cleaves trypsinogen to start activation cascade.
Bohr Effect
O2 binding and pH regulation. Low pH=more H+=more HbH+ bound=less O2 bound Indirect CO2 mechanism: More CO2=more H+=less O2bound
Unsaturated fatty acids
Omega is terminal carbon of tail Ex. Omega 6: 1st double bond is 6 carbons away terminus (linoleic acid)
Heme
Prosthetic group; porphyrin that chelates Iron with 2 His residues
Acid base Catalysis (ABC)
Proton transfer of sharing requiring side chain with proton. Ex. Glu, Asp, Lys, Arg, Cys, His, Ser, Tyr
Allosteric Inhibition
S-shaped with inhibitor. Glycolysis and phosphofructokinase.
Common fatty acid names
Saturated C12-Lauric acid C14-Myristic acid C16-Palmitic acid C18-Stearic acid Unsaturated C16-Palmitoleic acid (9th double bond from COO- end) C18-Oleic acid (9th double bond) C18-Linoleic acid (9 and 12 double bond)
Hemoglobin Affinity
Sigmodial; high affinity in lungs and low in tissues
Micelles
Single tailed (wedge-like) with Van der Waals forming micelles
Protein-ligand recognition
Size and shape of cleft & ligand
Carbonic Anhydrase
Speeds up CO2 to HCO3-(bicarbonate); enzyme contains Zn2+; Zn activates conversion of water to OH. Catalytic site is regenerated by release of products and binding of another H2O.
Michaelis Constant (Km)
Substrate concentration where Vo=1/2Vmax. Km is the dissociation constant for ES complex. Large Km= weak binding vice versa.
Induced Fit
Subtle changes to find favorable bound structure. Also has entropy reduction
T and R state
T (deoxy) vs R (oxy 4 O2 bound) state
Acyl-Enzyme intermediate
Tetrahedral intermediate collapses into relatively stable intermediate. His donates proton as general acid.
Micelles (cont)
Too big of micelle lead to hollow core that would fill with water. During flattening, water gaps still remain.
Cis vs trans
Trans is almost never produced naturally. found in artificial vegetable oils. Rigidifies hydrocarbon tail in extended conformation. Cis causes kink
Kinase
Transferase of phosphate group
Covalent Catalysis
Transient formation of ES covalent bonds can accelerate rates. Uses nucleophiles or electrophiles. Ex. Schiff base
Carbon Monoxide
Trapped in R State Allosteric regulation-CO locks into R state (blocking O2 release). 2-7hr half-life. 100% O2 treatment reduces to 10min half life
Microtubules
Tubulin. Kinesin (or actin/myosin V) flexible neck, motor protein.
Steady State
When there is much more substrate, ES formation is maximized and does not change.
Lipid monolayer
air-water interface like soap bubbles
Phospholipids (cont)
cis bonds cause kink Fats and oils are often saturated
Triacylglycerols (Fat storage in cells)
condensation of glycerol with 3 fatty acids stored as fats and oils, neutral at physiological pH
Gangliosides (complex sphingolipids)
found in brain membranes and protruding carbohydrates
Protease
hydrolase for amide bonds in protein
Accelerating Rxns
increasing Temperature or concentration of reactants (encounter rate)
Enzyme Turnover
kcat (catalytic constant)
Specificity Constant
kcat/Km good measure of catalytic activity larger #=compound is better substrate for enzyme
Lipid mobility
lipid tails are constantly in motion. Mobility is limited near head groups.
Glyolipids
membrane lipids with sugar head groups. 2 double bonds seperated by one methylene group
Ligand
molecule that binds to protein
Vitamin D
produced by UV light irradiating 7-dehydrocholesterol. 2 hydroxyl groups are added
Enzyme-catalyzed Reaction
reaction is 0 order at High [S]. 1st order at lower [S].
BPG (dynamic regulator of oxygen)
reduces O2 affinity; 2,3-Biphosphoglycerate No binding site in R and low affinity in T
Cholesterol
rigid ring structure alters membrane properties and is important to lipid bilayer
Sphingolipids
use sphingosine instead of glycerol as backbone; often H-bonded to another fatty acid through amide bond