biochem exam 3 written study (enzyme kinetics, inhibition)

what helps enzymes catalyze reactions?

-cofactors help enzymes catalyze reactions

-typically divalent cations (Mg2+, Fe2+, Zn2+)

what is a holoenzyme? an apoenzyme?

-holoenzyme: enzyme + cofactor = active

-apoenzyme: enzyme is inactive, no cofactor (ex: hemoglobin without heme)

what are coenzymes?

complex organic/organometallic cofactors (everything else that isn’t a metal ion)

what are enzymes and what do they bind?

-enzymes are proteins that do chemistry and bind to substrate

what is the difference between a coenzyme/enzyme and substrate?

-coenzymes/enzymes/cofactors are regenerated in catalytic cycle

-substrate is reacted and made into something new

what suffix indicates an enzyme? what do glycosylases and polymerases do?

-ase is a good indication something is an enzyme

-glycosylase: catalyze cleavage of glycosidic bond in DNA

-polymerase: catalyze polymerization of DNA

-some enzymes are named for their discoverer/function (ex: pepsin, lysozyme)

what do oxidoreductases do? hydrolases? lyases?

-oxidoreductase: transfer of electrons (hydride ions or H atoms) redox

-transferase: group transfer reactions

-hydrolases: use H2O to cleave

-lyases: cleave other types of bonds C-C, C-O, C-N

-isomerases: transfer of groups within molecules to yield isomeric forms

-ligases: formation of C-C, C-S, C-O, and C-N bonds by condensation reactions coupled to cleavage of ATP

what do ribozymes do?

-ribozymes are RNA enzymes (not all enzymes are proteins) that catalyze cleavage/ligation of nucleic acid

how is DNA synthesized? what does replicated DNA consist of? which enzyme helps with this?

-DNA is synthesized 5’ → 3’, DNA polymerase

-both strands of DNA must be copied before cell divides

-strand separates and each strand serves as template for new double helix

-daughter strand has complementary sequence to parent strand

-each copy of the replicated DNA consists of one parent strand and one daughter strand

what does the active site look like for DNA synthesis?

-protein interacting with the end of a duplex

-primer is the strand we are currently copying

-appropriate nTP

what is the substrate of DNA synthesis? what is the reaction?

-the appropriate NTP is the substrate

-in an SN2 reaction the 3’OH nucleophile attacks the alpha phosphate of the sugar (closest phosphate) and electrons end up forming a negative charge on pyrophosphate (PPi)

-end with DNA that is one nucleotide longer

what amino acid holds the cofactor?

-aspartic acid holds the metal ions

-metal ion cofactors improve nucleophilicity of 3’OH and improve leaving group, making the reaction better than it would be in free solution

what determines the next nucleobase added?

-the template strand determines the next nucleobase added

what are chemical kinetics? what is a collision model?

-chemical kinetics: how fast a reaction occurs → not about stability

-collision model: reactions are from particles colliding in right orientation with right amount of energy

what are factors affecting reaction rate?

-concentration (increases probability of collisions)

-temperature (increases energy to get to transition state, breaking bonds takes energetic input)

-structure + orientation (not all collisions are productive)

what is rate law?

aA + bB → cC + dD (where k exists over arrow)

-a is the number and A is the identity of molecule

rate = k [A]^a [B]^b

^^viewing rate as proportional to reactants

what are reaction orders?

-0th order: rate = k, independent of concentration

-1st order: rate = k [A] , depends on 1 reactant concentration

-2nd order: k [A][B] , depends on 2 reactant concentrations

in the collision model, what is the equation used and what do the variables mean?

k = Ae ^-Ea/RT

-A: frequency factor, describes how often reactants are colliding in correct orientation

-the exponential factor: fraction of molecules that have enough energy to react

-the Ea term is delta G double dagger

what is Ea? how does a catalyst change it?

-Ea (activation energy): minimum amount of energy required for a reaction to proceed

-Ea/delta G double dagger is always a barrier, nothing has no barrier

-catalysts lower activation energy (ex: catalyst for peroxide → water and O2)

why do we need catalysts?

-sometimes we only have so much reactant (concentration)

-increasing temp increases side reactions

-DNA is subject to 10,000 damage events per day

-the glycosidic bond of DNA must be broken to cut out an incorrect base (via glycosylases)

why can enzymes find the right portion of DNA faster than diffusion?

-if this is happening faster than diffusion this means it is not random

-random walk rate of diffusion: 10^8 M^-1 s^-1

-enzymes: 10^10 M^-1 s^-1

what are on the x and y axes of a reaction coordinate diagram?

-y-axis: free energy, G (amount of internal energy to do work)

-x-axis: reaction coordinate (molecular motion)

-read the reaction coordinate left to right

what is delta G ' standard?

delta G’standard is the standard free energy for biochemical reactions

-standard state defined only as pH 7.0

-affects the equilibrium constant

-delta G standard requires reactants to be at 1M, but that is not typical for biochemical reaction

what does delta G’ standard tell you? does it change with catalysis?

-delta G’ standard is thermodynamic

-tells you if reaction is spontaneous or not

-does not change with catalysis, is intrinsic to a reaction (all a catalyst does is affect ease of getting over hill)

thermodynamics do not change with catalyst, do kinetics?

kinetics can change with catalyst

what is delta G double dagger?

-delta G double dagger is activation energy

-activation energy is energy between the ground state and the transition state (top of hill)

-it is the minimum amount of energy required to break bonds needed to convert between substrate and product

what is a transition state?

-transition states are fleeting structures, that cannot be isolated unless trapped

what do enzymes do, change, not change? think K, delta G’ standard, delta G double dagger

-catalysts (enzymes) lower activation E, speed up rate of reaction

-enzyme is not consumed by reaction

-equilibrium (K) is not changed

-enzymes do not change delta G’ standard

-enzymes alter delta G double dagger (only affect kinetics)

how do enzymes work with the stickase model?

-no enzyme: substrate is metal stick

-transition state: bent stick

-products: broken stick

-enzymes favor the transition state of a reaction

why is the enzyme complementary to the transition state, not the substrate?

-if the enzyme substrate complex was complementary to the substrate, that would not help you get to the stick being broken

-the enzyme must be complementary to the transition state, and doesn’t bind the substrate or product strongly to stabilize (lower the energy) of the transition state via IMFs

what kinds of interactions exist between enzyme and substrate?

-some enzymes facilitate covalent interactions

-more commonly non-covalent interactions

what are the non-covalent characteristics of the active site?

-complementary shape (substrate fits in conformation of transition state)

-complementary chemistry (active site amino acids form stabilizing IMFs)

what is binding energy, delta GB

-there is some form of enthalpy (delta H) and entropy (delta S) for binding event

-contributes to enzyme specificity

-very important in lowering activation energy

how does entropy reduction from enzymes favor a reaction?

-collisions in solution can be rare and bringing two reactants together to form a single product is not entropically favorable

-binding free energy helps “pay” energetic cost of entropically restricting reactants

is delta S reaction more favorable when starting from a restrictive or nonrestrictive initial state?

-delta S of reaction is more favorable when starting from restrictive initial state (ex: chaperones + protein folding)

how does desolvation by enzymes catalyze reactions?

-start off with a bunch of water dissolving substrate (solvation shell), preventing collisions

-releasing a bunch of water has entropic benefit and allows substrate/enzyme binding

-enzyme substrate interactions replace substrate-solvent interactions

-destabilizing substrate increases reactivity

-higher starting E = smaller delta G double dagger/activation energy

how does distortion/alignment by enzymes catalyze reactions?

-enzymes help stabilize transition state structures, compensate for structural distortion of substrates needed to form products

-catalytic functional groups or prosthetic groups align to react with substrate, mediated by induced fit

how do the acid-base reactions by enzymes catalyze reactions?

-general acid base catalysis

-proton transfers mediated by amino acid residues in the active site

-make substrate/functional groups more nucleophilic (deprotonate) or electrophilic (protonate)

what did we see with the jolly rancher demonstration?

-generally you are able to do a certain amount of jolly ranchers per unit time but eventually you can’t do more (saturating)

does substrate concentration decrease or increase over time and how does this impact reaction rate?

-[S] decreases over time, which decreases the rate of the reaction

what is the initial rate of enzyme kinetics? what is happening early in the reaction?

-V0 is the initial velocity, this showed up as a linear dotted line in the slides

-early in the reaction [S] is relatively unchanged to product

-[P] , the amount of product versus time is linear

after this linear burst phase, things tail out at a Vmax and a ½ vmax. what exists at this ½ vmax on the x-axis?

-at ½ max there is Km

what exists at low [S], increasing [S] and higher [S]?

-at low [S], V0 increases linearly with the increase of [S]

-increasing [S] increases the probability the enzyme encounters substrate

-at higher [S] V0 stops increasing and a maximum V is approached as all enzymes have substrates bound

is the enzyme-substrate reaction zero, first, or second order?

-none! enzyme kinetics can’t be modeled so simply

-in the early phase of the saturation curve it is approximately 1st order and towards the end it is 0th order

so how do we model enzyme kinetics? what is pre-steady state and steady state?

-this type of enzymatic kinetics is modeled with the michaelis-menten equation

-pre-steady state: initial short period where the ES complex builds up

-steady state: rate is zero, an assumption, once complex has built up, get slow conversion from E+P to ES

what are the steps of the enzyme kinetics in the equation discussed?

-start with E+S

-then there is a k1 or on rate for the forward direction, and a k-1 for the backward direction moving to the next step

-enzyme collides with substrate and binds to form ES

-then there is a k2, a rate limiting, slow step and single headed arrow step

-end with E+P

*the binding is not rate limiting but the reaction is