Exam 2 Material Flashcards
Vo
- Initial velocity at the beginning of a reaction when the concentration of substrate greatly exceeds enzyme concentration
- Vo=Vmax[S]/Km+[S]
- dependent on the substrate concentration
Rate (velocity)
-Δ[A]/Δt or -Δ[B]/Δt or Δ[P]/Δt
High [S]
- 0 order, Rate=k, independent of [S]
- No increase in the rate of the reaction when more substrate is added
Low [S]
-1st order, rate increases with [S], rate=k[S]
Steady State Assumptions
- Concentration of substrate must be much greater than enzyme concentration
- ES remains constant (rate of formation equal to ES breakdown)
Km
- Michaelis constant
- Km=k2+k-1/k1
- Independent of substrate/enzyme concentration
- Inverse measure of how well a substrate binds to an enzyme (small Km=tight binding, large Km=weak binding)
- Km=[S] @1/2 Vmax (substrate concentration at one half of the max velocity=Km)
- Km low-reaches 1/2Vmax at lower [S] bc of higher affinity
Kcat
- The turnover number or catalytic constant
- How fast ES complex proceeds to E+P
- Equals the number of substrate molecules converted to product per unit time (=Vmax/total)
- 1St order rate constant (sec^-1)
Kcat/Km
- catalytic efficiency & specificity constant
- enzymes preference for different substrates
- Measures how enzyme performs when S is low
- Reflects binding and catalytic events, how the velocity changes according to how often E and S combine
- 2nd order (m^-1 S^-1)
Apparent Km
-measured value of Km in presence of inhibitor
Ki
- Dissociation constant for the inhibitor
- Measure of binding affinity
Competitive inhibitor
-Vmax stays the same but Km changes
Transition state analogs
- competitive inhibitors/compounds that resemble the transition state and block the active site
- bind much stronger to the enzyme than simple substrate or product analogs
- Cannot isolate transition state, just resemble
Noncompetitive inhibitors
- Bind reversibly to the enzyme
- Can bind, but wont form product
- Inhibitor binds to a site other than the active site
- Binding causes a change in the conformation of the active site so the substrate doesn’t efficiently form product
- Can bind to E or ES complex
Pure noncompetitive inhibitors
-Bind to a site far from the active site and do not affect substrate binding (apparent Km stays the same)
Mixed inhibitors
-Similar to noncompetitive except binding of substrate or the inhibitor affects the enzyme’s binding affinity for the other
-Two Ki’s- one for E and one for ES (different)
-Binds close to active site and alters both catalysis and binding
-Apparent Km increases, the inhibitor binds to a site close to active site & DOES decrease substrate binding
-Vmax decreases
(looks like competitive and noncompetitive)
Uncompetitive inhibitors
- Inhibitor binds to a site other than the active site but only when the substrate is bound (only binds to ES)
- Distorts active site, prevents reaction from occurring
- Apparent Km decreases, effectively increases affinity for the substrate
- Vmax decreases, the effects of un-competitive inhibition cannot be overcome by increasing [S]
- ES complex is constantly being depleted as inhibitor binds, producing ESI complexes
- Shifts E+S->ES equilibrium to the right toward more ES formation where it will bind more substrate to the enzymes to create more ES
- Leads to lower Km
Irreversible inhibitors
- inhibitors covalently modify the active site=permanent inhibition. Must wait for more enzyme to be made
- Ex: aspirin, nerve gas
Oganofluorophosphates
- Used as insecticides and nerve gas
- irreversible covalent inhibition of ACE by DIFP (an organofluorophosphate)
- phosphorous atom of VX covalently binds to a serine hydroxyl group in active site of ACE (VX gas and ACE has similar structures-competitive)
Atropine
- antidote to ACE inhibition, binds to acetylcholine receptors and acts as a competitive inhibitor in muscles
- more ACE is made to eventually regain control of the system
Induction
-Increase in amount/expression of transcript for enzyme produced caused by an effector molecule
Repression
-Decrease in amount or expression of transcript for enzyme produced caused by an effector molecule
Proteasomes
-Protein degradation by proteases in the lysosome or in macromolecular complexes
Zymogens
- Inactive precursor to an enzyme, activated by cleavage of a specific peptide bond
- inactive until they reach the proper environment (chymotrypsinogen to chymotrypsin and trypsinogen to trypsin)
Insulin
-synthesized as a precursor protein, modified to mature form by proteolysis
Proinsulin synthesized where? Transported where?
- In ER, oxidizing environment-folded and disulfide bonds formed
- Transported to the golgi apparatus, packaged into secretory vesicles, processed by proteases to form mature insulin
Reversible covalent modifications
- phosphorylation of Ser, Thr, Tyr (uses ATP) & usually occurs in response to a stimulus
- methylation of glu residues (used in bacteria as food sensor)
- creation or reduction of disulfide bonds
Allosteric enzymes
- Mostly multi subunit proteins with one or more active sites
- bind other ligands at sites other than the active site
- can be activated or inhibited by allosteric ligands, often control key reaction sin major pathways that must be regulated
- Michealis menten kinetics do not apply to allosteric enzymes
Vo vs [S] graph shape for allosteric enzymes
Sigmoidal
Activators
-positive modulators that bind to allosteric site and stabilize the active conformation, reaction rate increased
Inhibitors
-negative modulators that bind to allosteric site that stabilizes the inactive conformation, reaction rate decreased
Cooperativity
- Changes in the conformation of one subunit leads to conformational changes in adjacent subunits
- Can be positive or negative
Concerted allosteric model with activators and inhibitors
- all subunits are changed at once from taut (T) to relaxed (R) or vice versa
- activator shifts equilibrium in favor of the R, inhibitor shifts in favor of T
- S binds much tighter to R than to T
- cooperativity achieved because S binding increases the population of R, which increases the sites available to S
Homotropic effectors
-binding of substrate influences the binding of more substrate
Sequential Model
- Binding of the ligand to one subunit triggers a conformational change that is passed to subsequent subunits
- more complex model, allows for intermediate formations
Sequential Reactions
-reaction cannot proceed until all substrates are bound to the enzyme active site, ordered and random
Double-displacement reactions (ping pong)
- First product is released before second substrate binds
- Enzyme is altered by first phase of the reaction
Stabilizing the transition state
Lowers Ea and increases reaction rate
Catalysis promoted by proximity, orientation and strain effects
- enzyme brings substrate into close proximity with each other & catalytic groups (now not improbable collision of two molecules)
- enzyme binds substrates with a specific orientation that aligns the substrates and catalytic groups (now not improbable that molecules collide in correct orientation)
- enzymes freeze out transitional/rotational motion of substrates and catalytic groups
- upon substrate binding, transition state makes better contact with enzyme than substrate
Electrostatic Catalysis
charged enzyme functional groups in stabilizing otherwise unstable intermediates in the chemical mechanism
- substrate binds to enzyme, water is excluded from active site (desolvation)-protects reactive groups from water
- causes local dielectric constant to be lower-enhances electrostatic interactions in the active site(charge distribution helps position the substrate)
General Acid-Base catalysis
- proton transfer
- enzyme avoids unstable high energy charged intermediates by donating a proton (general acid) or accepting a proton (general base)
- if a group donates a proton, it has to get a different proton back by the end of a cycle
Covalent Catalysis
-form between a nucleophilic group on the enzyme and an electrophilic group on the substrate
Alkali metals role
-loosely bound and play structural roles
Transition metals
-usually play a functional role in catalysis as a part of a functional group
Metal Ion Catalysis
- Hold substrate properly oriented by coordinate covalent bonds, substrate held in very specific geometry so reaction can proceed efficiently
- enhances reaction by polarizing the scissle bond and stabilizing a negatively charged intermediate
Prosthetic groups
-coenzymes that ARE covalently bound to an enzyme and therefore are always present
epimers
stereoisomers that differ at only one carbon
enantiomers
stereoisomers that are mirror images (D&L)
Diasteromers
stereoisomers that are not mirror images
Anomers
stereoisomers that differ only at keto/aldo carbon
mutorotation
Interconversion between alpha and beta anomers through the open chain form
Starch Makeup
- amylose and amylopectin
- energy reservoir of plant cells & significant source of carbohydrate in the human diet
- found in chloroplasts of plant cells
Amylose
-repeating units of maltose-linear
Amylopectin
- Main backbone is amylose (linear) with D-glucose molecules in alpha 1->4 linkage
- has branches connect to backbone and to each other by alpha 1->6 linkages
- branch points every 25-30 glucoses
- has one reducing end
- has many non reducing ends
Glycogen
- Carbohydrate storage molecule in vertebrates (in liver and muscle cells)
- Liver: source of glucose for maintaining blood glucose
- Skeletal muscle: used to generate ATP during anaerobic muscle contraction
- starch-branching every 25 units, glycogen- every 8 to 10, ie more branching=short term storage, can lose all in 24 hrs
Branching
- allows several sites for simultaneous synthesis and degradation
- branching speeds up degradation
McArdles Disease
- Mutation in the muscle glycogen phosphorylase
- autosomal recessive, premature stop codon
- muscle cells can’t produce enough energy, so muscles become easily fatigued
Lipids
Substances from living things that dissolve in nonpolar solvents-insoluble in water
- storage molecules for energy
- gets lots of energy from fat
- stored in adipose tissue
amphipathic
have both hydrophilic and hydrophobic parts (fatty acids)
adipocytes
- only function is to store fat (lipids)
- found in oily droplets in the cytoplasm
- rich source of energy
Olestra
- Chemically synthesized fat (TAG) substitute
- Mixture of sugars and fatty acids
- not absorbed or metabolized, therefore not caloric
Saponification
- boil animal fat with lye (NaOH) to make soap
- NAS process
Orlistat
- treat obesity by inhibiting lipid absorption->reduce calorie intake-fat isn’t digested
- inhibits pancreas lipase
- side effect: oily and loose stool
Phospholipases 3 functions
-membrane remodeling, signal transduction, and digestion
Lysolethian
- One-legged phospholipids
- snake venom
- acts as detergent
- dissolves membranes in red blood cells which causes them to rupture
Sphingolipid
- polar membrane lipid, glycerol is replaced by sphingosine, long chained amino alcohol
- much more amphiphilic than tracylglycerols
Sphingomyelin
- found in most cell membranes, but most abundant in the myelin sheath of nerve cells
- insulates nerve axons
Glycolipids
- cerebrosides and gangliosides, abundant in the brain and nervous system membranes
- improper degradation results in metabolic disease
Tay-Sachs Disease
- Sphingolipid storage disease
- deficiency in B hexosaminidase A
- gangliosides accumulate in nerve cells, brain and spleen, lysosomes rupture
- results in blindness, weakness, seizures and mental retardation. Death usually by age 3
Gaucher Disease
Sphingolipid storage disease
-accumulation of glucocerebroside in lysosome in spleen, liver, kidneys, lungs, brain, bone marrow, white blood cells
-characterized by bruising, bone pain, fatigue, anemia, low blood platelets, and enlargement of the liver and spleen
IV enzyme replacement therapy effective
Cardiac Glycosides
- sugar derivatives of steroids
- increase cardiac muscle contraction
- can be toxic but also medicinally important (digitalis)
Hydropathy plots
-display the hydrophobic and hydrophilic regions of a protein sequence and predict the structure based on these regions
Plasma membrane is added to membrane by vesicles from
ER & Golgi
3 steps of membrane transport
-binding, change in shape of protein, release
Carrier proteins different from channel because
they move small numbers of molecules at a time
P glycoprotein
- ATP binding cassette transporter
- likely evolved as defense mechanism against harmful substances
- pumps out harmful xenobiotics
- protective function
- in blood brain barrier, precludes access of anti-cancer, anti-HIV, anti-psychotic and other therapies to the brain
- present in liver, small intestine and colon, limits the flow of drugs into the bloodstream
