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