MCB 2000 Exam 2

∆G < 0

• exergonic; spontaneous

• energy is released, the reaction can proceed as written

∆G = 0

• equilibrium; no net change

• forward and reverse rates are balanced. concentrations stay put

∆G > 0

• endergonic; non spontaneous

• energy must be supplied for the reaction to proceed forward

∆H

• enthalpy change

• heat absorbed or released. negative = bonds in products are stronger than bonds in reactants

• favors spontaneity

T

• temperature

• always in kelvin. acts as a multiplier on ∆S, scaling how much entropy matters at this temperature

∆S

• entropy change

• disorder of the system

• +∆S = more disorder in products

• favors spontaneity ( - sign flips it)

what ∆G can tell you

• whether the reaction can proceed as written

• which direction is favored at the moment

• how much energy is released/required

what ∆G doesn’t tell you

• how fast the reaction will go

• whether an activation barrier is in the way

• what the reaction looks like inside the cell

∆G˚’

• standard

• measured in standard conditions: 1M for all reactants and products, 25˚C, pH 7 (the prime mark)

actual ∆G

• what’s happening right now in the cell

• the actual concentrations of reactants and products at this moment

• the number that decides whether the reaction proceeds

Q

[products]/[reactants]

Q small

• lots of reactants, few products

• reaction wants to go forward

Q = Keq

• concentrations match equilibrium

• no net change. ∆G = 0

Q large

• lots of product, few reactants

• reaction wants to go backwards

∆G˚ < 0 then Keq < 1

• products win

• equilibrium sits to the right. the forward reaction is favored at standard conditions

∆G˚’ = 0 then Keq = 1

• evenly split

• reactants and products at equal concentrations at equilbrium

∆G˚’ > 0, then Keq < 1

• reactants win

• equilibrium stays to the left. most of the material stays as reactants at standard conditions

enzymes

biological catalysts; proteins that make reactions go without being consumed

substrate

starting material; the molecule the enzyme acts on

enzyme-substrate complex

a short-lived intermediate where substrate is held in the active site

product

what you get out. the enzyme releases it and it’s ready to bind again

∆G

the energy difference between substrates and products is set by their structures, not by the enzyme

Keq

the position of equilibrium doesn’t move. the enzyme reaches it faster, but lands in the same place

X キ(transition state)

• no longer substrate, not yet product

• a high-energy, unstable structure with bonds half-broken and half-formed. the hardest step on the path

∆G キ (activation energy)

• the energy needed to reach Xキ

• high ∆Gキ means a slow reaction. low ∆Gキ means a fast one. this is the barrier enzymes work on

the active site

a small, specifically shaped region where the chemistry actually happens

active site is a 3D cleft

a pocket, groove, or crevice on the enzyme surface, shaped to receive a particular substrate

active site is a small fraction

only a few residues out of hundreds. most of the enzyme is scaffolding holding those residues in place

active site from residues come from far apart

active-site residues may be scattered across the primary sequence but brought together by folding

trypsin

• digestive

• cleaves peptide bonds

• after Lys and Arg

• small intestine; cleaves dietary protein into smaller peptides

thrombin

• blood clotting

• cleaves peptide bonds

• after specific Arg residues only

• in the bloodstream; cleaves fibrinogen at exactly the right place to form clots

lock and key

• the active site is pre-formed; the substrate fits as it is

• rigid complementary shape

• substrate slips in like a key and lock

• simple but doesn’t capture how most enzymes really work

induced fit

• the active site reshapes around the substrate as it binds

• active site is flexible, not pre-formed

• substrate triggers a conformational change that closes the site around it

cofactors

chemistry helpers; some enzymes need a non-protein partner to do their job; coenzymes and metal ions

coenzymes

organic molecules; often vitamin-derived. ex: NAD⁺ from niacin, FAD from riboflavin, coenzyme A from pantothenic acid

metal ions

inorganic ions; ex: Zn²⁺ (carbonic anhydrase), Mg²⁺ (kinases, polymerases), Fe²⁺ (cytochromes)

prosthetic group

a cofactor (either type) that’s tightly, often covalently, bound to the enzyme

six classes of enzymes

• oxidoreductase

• transferases

• hydrolases

• lysases

• isomerases

• ligases

oxidation-reduction

move electrons between molecules

transferases

move functional groups from one molecule to another

hydrolases

cleave bonds by adding water

lysases

break or form bonds without water or redox; often make double bonds

isomerases

rearrange functional groups within a single molecule

ligases

join two molecules at the cost of ATP

michaelis-menten equation

• one equation describes the entire hyperbolic curve

• V_o = V_max [S]/(K_m + [S])

when [S] < K_m

• V_o = (V_max/K_m) * [S]

• linear in [S]. rate scales with substrate

when [S] = K_m

• V_o = ½V_max

• _½ saturation point. the definition of K_m

when [S] > K_m

• V_o = V_max

• saturated. adding substrate doesn’t

V_max

• maximum rate; reached when every enzyme molecule is bound to a substrate at any given moment

• the enzyme is the bottleneck

• at V_max adding more substrate does nothing. enzyme is already working flat-out

• to go faster, the cell has to make more enzyme

• V_max is proportional to [E]_T, the total enzyme concentration is the assay

K_m

• ½ saturation point

• K_m = [S] at which V_o = ½ V_max

• units is M

low K_m

• high affinity

• enzyme reaches ½ V_max even when [S] is small. tight binding. works well at low substrate

[S] > K_m

• underused

• rate is well below V_max. the cell built enzymes that mostly sit idle. could be running faster with the same machinery

[S] = K_m

• roughly ½ saturated

• already at V_max. more substrate gives no more rate. cell paid to make and ship a substrate it can’t use faster

kcat

• the turnover number; how many substrate molecules each enzyme processes per second

• kcat = V_max/[E]_T

• units in per second

catalytic efficiency

• kcat/K_m

• single best number for comparing enzymes

• units of M^-1s^-1

• measures speed x specificity in one number

high kcat/K_m

fast and good at finding it’s subtrate

catalytically perfect

kcat/K_m approaches 10⁸-10⁹ M^-1s^-1. limited by only by how fast substrate can diffuse to the active site

diffusion limit

10⁸-10⁹ M^-1s^-1; as fast as substrate can possibly arrive

finding V_max

• V_max = 1/y-intercept

• read where the line crosses the 1/V_o axis

• take the reciprocal → V_max

• sign → always positive

finding K_m

• K_m = -1/x-intercept

• read where the line crosses 1/[S] axis. this value is negative

• take the reciprocal, then negate it. that’s K_m

• sign → K_m is positive once the negative is applied

what L-B is good for

• quick visual sanity check on a dataset

• easy to spot outliers (stuck points jump off the line)

• clean, direct readouts for V_max and K_m from intercepts

• standard way to teach how inhibitors change parameters

what L-B isn’t good for

• reciprocals at low [S] become huge → they dominate the fit

• measurement noise gets amplified at the low-[S] end

• linear regression weighs all points equally, but they aren’t equal

• modern drug screening uses nonlinear fits to the original M-M curve instead

homotropic effect

substrate itself acts as a regulator; same molecule, two roles

highly cooperative allosterics

• ratio = 4

• [S] only has to change 4-fold to go from 10% to 80%. sharp, switch-like response

M-M

• ratio = 27

• [S] must change 27-fold to go from 10% to 80% saturation. show, gradual response

M-M enzymes

• workhorses

• hyperbolic kinetics, single subunit

• rate scales smoothly with [S]

• drive the bulk chemistry of the pathway

allosteric enzymes

• switches

• sigmoidal kinetics, multimeric

• switch-like response to small [S] changes

• sit at pathway control points; tunable by activators and inhibitors

two aldehyde dehydrogenase isozymes

same reaction, different K_m values. each one has a different job

low K_m isozyme

• workhouse

• high affinity. binds acetaldehyde even at very low concentrations

• handles the bulk of acetaldehyde clearance under normal conditions. keep blood levels low

high K_m isozyme

• backup

• low affinity. only kicks in when acetaldehyde is already very high

• designed for surge capacity, not baseline clearance. can’t keep up if the low-K_m form is broken

alcohol flush

a mutation inactivates the low-K_m isozyme. the high-K_m alone can’t keep up

mutation (alcohol flush)

inherited variant inactivates the low-K_m ALDH. common in East Asian populations

acetaldehyde piles up (alcohol flush)

high-K_m ALDH alone has too low an affinity to clear acetaldehyde at normal levels. it accumulates in the blood

symptoms (alcohol flush)

acetaldehyde causes the flushing, rapid heartbeat, and nausea characteristic of alcohol flush reaction

PRS is regulated

phosphoribosylpyrophosphate synthetase sits at the start of purine synthesis. allosteric inhibitors slow it down when purines are abundant

regulation lost

a mutation breaks the allosteric site. inhibitors can no longer bind. PRS runs flat out regardless of cellular need

urate accummulates

excess purines are degraded to urate. urate crystallizes in joints. the result is gout: painful inflammation from a kinetic failure upstream

how kinetics shape pathway flux at the enzyme level

how much enzyme is around. V_max scales linearly with [E]_T

how kinetics shapes pathway flux at the substrate level

where on the M-M curve the cell sits. [S] near K_m gives smooth control

how kinetics shapes pathway flux at allosteric tuning

activators and inhibitors at non-active sites shift sigmoidal curves left or right

four strategies to lower activation energy

• covalent catalysis

• general acid-base

• metal ion catalysis

• approximation

covalent catalysis

an active site nucleophile briefly forms a covalent bond with the substrate, opening a faster path to product

general acid-base

a residue donates a proton at just the right moment, stabilizing the transition state

metal ion catalysis

a bound metal stabilizes negative charge, polarizes a bond, or acts as an electrophile

appro