Unit 2 Protein Function Cell Bio

reaction equilibrium

K_eq= K_forward/K_reverse = [products]/[reactants] (constant)

binding reactions

formation of intermolecular complexes is an equilibrium reactions; molecules bounce into each other randomly; noncovalent bonds stabilize the interaction

association and dissociation rates

each binding reaction has a unique…

never zero

the dissociation rate is ….

Kon and Koff

non-covalent bonds determine these

forward or reverse

every reaction can occur in the ______ or ______ directions

converge

reaction systems will _____ on their equilibrium

energy

moving reactions away from equilibrium requires ______; the further reactions are away from equilibrium, the more ______ is required or released

Gibbs Free Energy

change in this measures how much energy would be released by a reaction to occur given reagent concentrations

makes no statement about reaction rate

ΔG = 0

the reaction is at equilibrium

negative ΔG

reaction is exergonic, releases energy, and can proceed spontaneously

positive ΔG

reaction is endergonic, requires energy input, and cannot proceed spontaneously

move a system away

ΔG_forward=-ΔG_reverse

accounts for energy needed to ____________ from the equilibrium

absolute energy state

cannot calculate this before or after a reaction, only the change in free energy (ΔG)

how to calculate Gibb’s free energy

ΔG^o’ measured under defined “standard conditions”: all products and reactants are 1 M, pH=7

ΔG^o’ is specific to each reaction

how to make endergonic reactions proceed

1. ΔG for a reaction is concentration-dependent

– log ([Products] / [Reactants]) is a measure of actual reaction conditions and can be negative

– may offset a positive ΔG°’

2. Link an exergonic reaction to an endergonic reaction, so that the overall ΔG is negative

(ΔGs for sequential reactions are additive)

ATP (adenosine triphosphate)

main energy carrier in a cell; hydrolyzed to ADP (adenosine diphosphate) with release of energy; one constituent of RNA

concentration dependent

ΔG is…

cellular steady state

is not the same as chemical equilibrium; ideal state for a cell to survive and perform the necessary functions

linking reactions

exergonic to endergonic so that overall ΔG is negative

free energy summary

• ΔG is the change in free energy for a reaction

• only reactions with a negative ΔG can proceed spontaneously

• ΔG is highly dependent on molecular concentration inside cells

• ΔG for sequential reactions is additive reactions with a positive ΔG°’ can proceed if:

– concentrations of reactants or products are changed, so that ΔG is negative

– coupled to a reaction with negative ΔG

• ΔG makes no statement regarding reaction rate.

high energy transition state

Reactions require overcoming a __________ ________ before a reaction can proceed

sufficient

only a small fraction of molecules have _______ energy

enzymes

can lower the energy requirement significantly, thereby increasing the rate of the reaction

starting and ending energy levels

the number of transition steps and their energy requirements are irrelevant, ΔG is only determined by…

three different ways for enzyme catalysis

A) enzyme binds to two substrate molecules and orients them precisely to encourage a reaction to occur between them

B) binding of substrate to enzyme rearranges electrons in the substrate, creating partial negative and positive charges that favor a reaction

C) enzyme strains the bound substrate molecule, forcing it toward a transition state to favor a reaction

orients

enzyme binds to two substrate molecules and _______ them precisely to encourage a reaction to occur between them

rearranges electrons

binding of substrate to enzyme _______________ in the substrate, creating partial negative and positive charges that favor a reaction

forcing it toward

enzyme strains the bound substrate molecule, ______________ a transition state to favor a reaction

steps in enzyme catalysis

1. enzyme binds substrate, forming E-S complex – via non-covalent bonds – E and S meet randomly

2. catalysis happens, E-S complex becomes enzyme-product complex (E-P)

3. enzyme and product dissociate

   Enzymes accelerate rate of reaction,

   cannot change K eq!

lysozyme

takes a six-sugar molecule (a hexasaccharide) and cleaves it between sugars 4 and 5.

conformation

lowered activation energy because enzyme twists sugars into ___________ resembling activation state

covalent bond

a _________ linking enzyme and substrate forms briefly during catalysis (not true for all enzymes)

reconstitutes

release of product ________ enzyme

noncovalent bonds

enzyme and substrate bind through _____________ (key/lock analogy or induced fit)

higher energy state

some enzymes twist substrate into a _________________, resembling the reaction intermediate

lowers

enzymes ______ the activation energy for the reaction, so more molecules can react

→ enzymatic reaction occurs (faster than unassisted) → enzyme and product dissociate

catalyst

enzymes as _________: must return to their starting state

simultaneous hydrolysis

may meet energy requirement by ____________of high-energy molecules, eg. ATP

protein concentration (levels)

transcription, translation, destruction, and localization

conformational change

activity of protein regulation: regulated transition between active and inactive states

allosteric activators/inhibitors, posttranslational modifications, and complex formation

ways for conformational change to occur

substrate availability and binding to active site

sequestration of substrate; and competitive inhibitors compete with substrate for binding

positive feedback

example: as the concentration of ADP increases, the conversion of food molecules into ATP is increased, activating the reaction

arrowhead

negative feedback

end products of biochemical pathways will inhibit an early step in the pathway (product inhibition), shutting it down

square line

allosteric inhibition

binding usually not at active site, but at a regulatory site

inactivating

binding causes conformational change, _________ enzyme

EF-Tu protein example

switches between active and inactive states; regulated by a small molecule → induces large-scale conformational change

• bound to GTP: red domain is -helix, binds tRNA → EF-Tu is active

• bound to GDP: red domain is a loop, no tRNA binding → EF-Tu is inactive

allosteric activation

Binding of activator causes conformational change and the binding of activator and substrate is cooperative.

allosteric inhibition

Binding of inhibitor causes conformational change, which is incompatible with substrate binding.

multi-subunit inhibition

addition of first inhibitor is energetically unfavorable → forces change in additional subunits and breaks the symmetry

additional inhibitors bind more easily → restores symmetry and there is cooperative binding

response to inhibitor is much steeper

more difficult; entire complex

in multi-subunit inhibition, ___________ to bind first inhibitor, but additional inhibitors bind more easily. Once the first inhibitor binds, the __________ is affected

on/off switch

Inhibition is more rapid and more closely resembles this when there are more and more subunits

competitive inhibition with ATP

ATP-γS is a non-hydrolysable analog of ATP, and its overall shape of molecule is unchanged: expected to bind with similar characteristics as ATP

ATP-γS competes with ATP for binding to the active site → γS strongly inhibits hydrolysis of phosphate ester

allosteric inhibition summary

molecule binds away from the active site, the enzyme conformation is changed, and it affects the rates of substrate binding and processing

competitive inhibition summary

molecule binds at active site directly, enzyme conformation is unchanged, competes with substrate for binding, and the inhibitor is usually chemically inert

competitive or allosteric inhibitor example

LQM is an allosteric inhibitor because it shut down any and all reactivity; increasing the inhibitor meant no product was formed; when there were equal amounts of enzyme and inhibitor, some product was made, and with more inhibitor, regardless of the amount of substrate, zero product was formed.

ratios

competitive: increase the amount of substrate = increase in product

allosteric: increase amount of enzyme = decreased inhibition

inhibitor to substrate

ratio that matters for competitive inhibition

overwhelm inhibitor, with some substrate, means product will form

concentration of inhibitor

A factor that matters for allosteric inhibition

With too many binding sites, some enzymes without the inhibitor can process substrate into product;

not dependent on substrate concentration

Posttranslational Modifications

enzymatic addition of groups to polypeptide chains by covalent bonds; they affect: protein shape, ionic charge, protein stability, protein-protein interaction, enzymatic activity, and subcellular localization

common modifications

phosphorylation, methylation, acetylation, prenylation, ubiquitination, sumoylation, glycoslyation

fast and reversible

posttranslational modifications occur because they are …

kinase

enzyme that adds phosphate

phosphatase

enzyme that removes phosphate

phosphorylation

kinase modify specific amino acids: tyrosine, serine, and threonine.

The enzymes kinase and phosphatase add/remove negative charges, and this process is key in cell signaling

tyrosine phosphorylation

kinases transfer the γ-phosphate from ATP

inactive

some proteins become ______ by phosphorylation

Src kinase

part of a virus that causes cancer in chickens; the viral gene is a copy of the human gene that lacks the c-terminal inhibitory tyrosine

During phosphorylation, the phosphate removal loosens the structure, the activating ligand binds to the SH3 domain, and the enzyme can now phosphorylate tyrosine to self-activate

signal integrators

Proteins can serve as _________

• Are certain residues phosphorylated?

• Are certain residues unphosphorylated?

• Are certain binding partners present?

• Are certain binding partners absent?

highly regulated

protein activity is _________ through positive and negative feedback

regulation

can be allosteric or competitive

– allosteric regulators bind away from the active site of the protein

– Competitive regulators bind to the active site

Conformational change can regulate protein activity

reversibly

protein activity can be ________ regulated by post-translational modifications

phosphates

added by kinases and removed by phosphatases