define energetics

can tell you if a reaction will go on its own or if you need to put energy into it to make it happen (how far a reaction will occur) or be spontaneous on its own based on amounts present in test tube or in a cell

define kinetics

can tell how fast a reaction can occur, how much substrate is needed to catalyze a reaction, and whether/how another molecule is inhibiting the enzyme

what do enzymes do and how

- catalyze reactions, speeding them up 10^6 - fold or more - enzymes do not change the equilibrium of the reactions - enzymes DO change the activation energy of the reaction - they are highly specific binding molecules based on a precise interaction b/w the substrate molecules and enzyme - enzymes stabilize the transition state

two types of models of enzymes binding to substrate

speeds up interactions and very picky about who they interact w/ chem reaction= reactant catalyze reaction: substrate - lock and key model the substrate (key) fits perfectly in the enzyme (lock) which allows reaction to occur - induced fit model, squishy model the substrate (glove) fits better as the enzyme (hand) binds to it.

measuring the energetics of an uncatalyzed reaction

at 30 minutes, this reaction has gone to equilibrium and the quantity of C and D does not change

basic enzyme energetics thought reaction equation and at equilibrium Keq equation and gibbs free energy change equations

A+B double arrows C + D Keq= [C][D]/[A][B] the energy released (or required) based on the equilibrium concentrations of reactants and products is calculated by triangle G= -RTln[C][D]/[A][B] or triangle G= -RTlnKeq

What does R stand for in gibbs free energy change and what does T stand for

R= universal gas constant T= temperature in kelvin or absolute temperature

exergonic, endergonic, and equilibrium reaction

Keq= greater than 1, triangle G= - , exergonic, a little bit of C & D Keq= less than 1, triangle G= +, endergonic, a lot of C & D Keq= 1, triangle G= 0, equilibrium, C+D=A+B

what type of equation is when reactants have more energy than products

very little A + B Keq >> 1 delta G knot << 1 exergonic more energy in A and B than C and D

how to measure the energetics of a catalyzed reaction

1. time=0 add compounds A and B and mix well, E= enzyme is present 2. 0.06 second sample and measure amount of C and D 3. repeat at 0.12 sec 4. at 0.18 sec repeat enzyme catalyzed reaction is complete, 10^6 fold rxn

give equations for delta (triangle) G

Overall energy released or required is calculated by delta G knot + RTln [Ci][Di]/[Ai][Bi] or delta G knot + RTlnK C+D= Products (top) A+B= Substrates

delta G definitions

Gibbs free energy change, is the free energy change of a rxn and depends on the concentrations of reactants and products

if delta G is negative, if delta G is positive how can the rxn proceed?

delta G is negative, rxn can proceed with spontaneously with release of energy (exergonic), delta G is positive: rxn requires input of energy to proceed with endergonic, rxn not likely will occur

Delta G knot definition

standard free energy of a reaction when reactants and products are initially at 1M concentration, 1 atmosphere pressure and 25 C (298 K), MORE APPLICALE TO CHEMICAL RXN

Delta G knot prime definition

standard free energy change at pH 7, 1 atmosphere pressure. More applicable to biochemical rxns, 298 K

summary of presentation 2: thermodynamics prt 2

- enzymes do not change the final equilibrium of a rxn - enzymes change the rate of a rxn - the standard free energy change of a rxn delta G knot= -RTlnKeq or standard free energy change at pH 7 delta G knot prime= -RTlnKeq, depend on the Keq - free energy change of a rxn delta G depends on the concentrations of reactants, products, and temp, and may be calculated by the formula delta G=delta G knot prime +RTlnK - whereas the delta G knot and delta G knot prime, describe a completed rxn the delta G describes free energy of a rxn at any pt before reaching equilibrium

define transition state

transition state is that point at the top of the energy hill where the rxn could go forward or backwards, back to substrate or forward to product

transition state in uncatalyzed rxn

to get to transition state we need to add some energy -> energy hill substrate aka ground state net delta G is that from the ground state to the product state delta G behaves like a rock rolling down a hill - images on slide

transition state in a catalyzed rxn

enzymes lower the energy hill, or activation energy. Allowing the rxn to proceed with less energy input - look at images on slide, be able to draw the graph

what does enzymes active sites use to bind to the substrate? and what do they form?

the enzyme active site has high specificity for bindings its substrate to form an enzyme-substrate complex - the binding of substrates use many weak interactions in the active side ex: lock and key and induced fit are two models for substrate binding - might have displacement of water = gain entropy, or H bonds or van der waals, might form ionic/salt bridges

how do enzymes reduce the activation energy and bind substrates

-substrate binding can release enough energy that is used to overcome the activation energy -when the substrate binds to the active site of the enzyme, it does not fit exactly right, thus forcing the substrate into its transitional state structure ex: drugs, resembling the transition state, and they are typically called transition state analogs

what is enzyme kinetics and what it tell me

how fast a rxn occurs how much substrate is needed inhibition of the enzyme if there is allosteric regulation order of substrate binding (can have 1 or more substrates binding with a product)

how to measure rate of a catalyzed rxn

add substrate S and E (enzyme) mix well, start with a low concentration of S then keep repeating the experiment using higher and higher substrate concentrations measure at 60, 120, and 180 seconds, measuring product amount being made

measuring rate of a catalyzed rxn after info has been taken step 1 and 2

1. plot the data - plot product concentration vs time - measure the slop of plot to determine Vo - at low [S] the enzyme is not saturated by substrate, so the rxn rate is less than maximal 2. replot initial velocity vs substrate concentration

define Vmax Km Kcat

-vmax= max rate of enzyme catalysis when saturated with substrate, -Km, michaelis-menten consntant, is the substrate concentration at 1/2 Vmax, how much molecules enzymes turn over per second or ms Kcat: or turnover number is the number of substrate molecules used or converted by an enzyme per unit time when fully saturated

michaelis-menten equation

V0= Vmax ([S]/[S] + Km) substrate presence increases= velocity increases

lineweaver-burk plot

this transformation allows plotting of enzyme kinetics data on a linear plot 1/Vo = (Km/Vmax)(1/[s] +1/Vmax)

what are the two types of enzymes

unregulated: always on allosteric: catalytic activity can be regulated

how are metabolic pathways controlled

allosteric enzymes are multi-subunit proteins that might have a quaternary structure composed of several different peptide chains allosteric enzymes typically bind effector molecules at regulatory sites distinct from the substrate binding site for catalysis allosertic enzymes have non-michaelis-menten kinetics allosertic enzymes are found at committing steps in metabolic pathways

where are allosteric proteins found

- committing steps to pathways - at beginning mostly found at beginning and branch points-

types of regulatory molecules with allosteric proteins

positive: turn enzymes on negative: turn enzymes off I= negative regulatory molecule A= positive regulatory molecule

two states of allosteric proteins

T= tensed state, catalytically inactive form R= relaxed state, catalytically active form

how do regulatory molecules and allosteric proteins work

in solution, allosteric proteins primarily exist in the T (OFF) STATE binding of regulatory molecules or substrates stabilizes the T or R state - a negative regulatory molecule stabilizes the T state "off" - a positive regulatory molecule stabilizes the R state "on"

list two models for conformational changes in allosteric proteins

MWC model sequential model

MWC (monod-wyman-changeux) or concerted change model

- allosteric proteins are multi-subunit proteins with effort and substrate binding sites on each subunit - binding of a substrate or effector molecule stabilizes the R form of a subunit, stabilizing the R form for all the subunits - this is an all or none model where all the proteins subunits are in the R state or T state

sequential model

- binding of effector or substrate molecule stabilizes 1 subunit in the R state - bound subunit influences the stability of an adj subunit that will subsequently be stabilized in the R state on binding of a substrate or effector molecule - all or none model, since there can be forms of the protein with 1, or 2, or more subunits stabilized in the R state

how can enzymes inhibit

- reversible: where an inhibitor can diffuse away from the enzyme - irreversible: where an inhibitor covalently attaches to the enzyme, essentially killing the enzyme

what can enzymes inhibit

typically inhibitors are molecules that 1. chemically resemble the natural substrates for an enzyme 2. chemically resemble the transition state of a enzyme bound substrate 3. chemically resemble a regulatory or effector molecule

where can enzymes inhibit

inhibitors can bind - at the enzyme active site - away from the active site, for example, at regulatory sites in allosteric enzymes

how do competitive inhibitors work in reversible enzyme inhibitors

competitive inhibitors work by competing with the natural substrate: the higher the concentration of the inhibitor, the more likely the probability that the enzyme will choose to bind it rather than to the substrate or vice versa Ex: yellow M&MS/brown - we can use enzyme assays with and w/o inhibitors to determine the kind of inhibitor ex: double reciprocal plot or MM plot

what is an example of competitive inhibitor?

ibuprofen inhibits prostaglandin synthesis

noncompetitive or mixed inhibition: reversible enzyme inhibitors

noncompetitive inhibitors can bind an enzyme alone or an enzyme that's around bound to a substrate these inhibitors work by inhibiting the enzyme activity directly prevent proteins from catalyzing

uncompetitive inhibition: reversible enzyme inhibitors

uncompetitive inhibitors work by binding at a different site to the substrate uncompetitive inhibitors bind to the ES complex these inhibitors work by inhibit the enzyme activity

irreversible inhibitors

irreversible inhibitors covalently modify the enzyme effectively killing the enzyme activity. suicide substrate inhibits also work same mechanisms

transition state analogs

molecules that resemble the TS of a substrate being catalyzed to product Fdump is a TS analog for the enzyme thymidylate synthase FdUMP is also a suicide substrate or irreversible inhibitor