Enzyme Kinetics Pt. 1 Flashcards
Functions of Enzymes
- higher reaction rates
- milder reaction conditions
- greater specificity
- capacity for control
Active Site
- region of an enzyme binding the substrate
- usually clefts/crevices in protein
- amino acids and cofactors are held in precise arrangement with respect to substrate structure
- amino acids in active site define specificity
Lock and Key Model
- ligand binding site is rigid and complementary to ligand shape
Induced Fit Model
- flexible interaction between ligand and active site induced conformational change (adaptation leads to perfect fit)
- enhances reaction mechanism and enables more specific fit
- stabilises transition state
Substrate Specificity
- geometric specificity = active site complementary to structure of substrate
- electronic specificity = amino acids in active site interact with substrate so that only the substrate can favorably bind to form the enzyme substrate complex
Geometric Specificity
- selective about chemical groups of the substrate
- more stringent requirements
- varying degrees of geometric specificity
- few enzymes are absolutely specific for one substrate
- some work on a group of related molecules
Electronic Specificity
- opposite charges attract
- hydrophobic and hydrophilic attractions
Stereospecificity
- enzymes are highly specific in binding chiral substrates and in catalysing their reactions
- discriminates between enantiomeric substrates
- stereospecificity is due to enzymes active site
- absolute stereospecificity
CIP Rules for Chirality
- orient lowest priority group (lowest atomic number) facing away
- number 3 groups by increasing priority
- determine rotation of groups in decreasing priority
- clockwise: R
- counterclockwise: S
Oxidoreductases
redox reactions, catalyse H+ and O atom transfer
Transferase
transfer of functions groups to other compounds
Hydrolyses
catalyse the hydrolytic cleavage of C-O, C-N, C-C and some other bonds like anhydride and phosphoric groups
Lyases
cleave C-C, C-O, C-N bonds by elimination, cleaving double bonds or rings or adding groups to double bonds
Isomerases
catalyses geometric/structural changes in a molecule (isomerisation)
Ligases
catalyses joining of 2 molecules couple with hydrolysis of diphosphate bond
Cofactors
- some enzymes require small molecules during catalysis
- cofactors are metal ions/organic molecules
- organic molecules: coenzymes
- cosubstrates: behaves like substrate and leaves after the reaction
- prosthetic group: tightly bound and remains after reaction
Vitamins as Coenzyme Precursors
- many organisms are unable to synthesise parts of essential cofactors
- substrates are present in organisms diet
- vitamins that are precursors are water soluble
Chemical Kinetics
- study of reaction rates
- binding affinities
- enzyme mechanism
- influence of conditions on rate
Rate of Reaction
Proportional to frequency of reaction molecule collision
rate = k [A]^a[B]^b
Reaction Order
Power dependance of rate on concentration of species involved
Sum of powers in rate equation
1st order: one molecular changes to each other
2nd order: two molecules reacting
1st order reactions
- direct proportionality between rate and concentration
- natural log of concentration is directly proportional to time (straight line down)
2nd order reactions
- rate proportional to the square of concentration of one reactant
Half-Life
time for substrates concentration to fall to 1/2 their initial value
- 1st order half life = ln2/k
- 2nd order half life = 1/k[A]o
- independent of inital substrate concentration
Rate Determining Step
- slowest intermediate step in the reaction that determines the rate of the total reaction
- RDS determines the overall rate equation
Collision Theory
- properly oriented
2. sufficiently energetic collision
Boltzmann Maxwell Graph
Average kinetic energy of molecules is proportional to absolute temperature
Higher temperature means more molecules have KE greater than or equal to Ea
Arrhenius Equation
k = Ae^-Ea/RT
A = pre exponential/frequency factor is fraction of sufficiently energetic collisions that lead to reaction
Ea (kj/mol) = minimum amount of energy needed for a reaction to occur upon collision
Arrhenius Graph
ln k = ln A - Ea/RT
slope = -Ea/R
y intercept = ln A
high activation energy = steeper slope at a rate sensitive to temperature
Transition State
Enzymes enhance the rate of reaction by stabilising the TS of the reaction. Active site is more favorable to the transition state so the substrate is forced to adopt this.
TS is a metastable compound sharing features of both the reactants and products
Some reactant bonds are being broken while product bonds are being formed
NOT the same as the intermediate state and not able to be isolated or purified
Erying Equation
Transition state is in rapid equilibrium with reactants and eq constant K (dagger)
Enzymatic Rate of Reaction
Rate lowered by lowering the activation barrier between reactants/transition state, increasing fraction of reactants able to achieve the TS
Kinetic barrier lowered to the same extent for forward and reverse reactions as ground state of free substrates/products remain the same
Catalyst doesn’t affect position of equilibrium
Reducing the Activation Barrier
By forming H bonds or hydrophobic contacts, enzymes are increase rate by 10
- stabilising transition state
- destabilising ground state of enzyme bound substrates and products
- introduces how reaction pathway with a different TS with lower free energy
- initial interaction is noncovalent and uses H bonds, hydrophobic interactions, VDW forces etc
- favorable interaction between E and S results in a favorable intrinsic binding energy
- there is a loss of entropy as two species become one, and the substrate is more ordered
Transition State Analogues
- have high affinity for the active site, higher than the substrate as they are an exact fit
- act as inhibitors
- used to elucidate mechanism of reaction
Catalytic Antibodies
- antibodies tightly bind their antigen but without altering its chemical nature
- theoretically, if an antibody binds a TS molecule it may be expected to catalyse a corresponding chemical reaction by forcing substrates into the TS geometry
Hammond Postulate
If 2 states (eg. TS and unstable intermediate) occur consecutively during a reaction process and have nearly the same energy content, their interconversion will involve only a small reorganisation of the molecular structures
Species that are sequential on the reaction coorinate + similar in energy are similar in structure
- Exothermic reaction: TS resembles reactants
- Endothermic reaction: TS resembles products
Desolvation
- substrate binding to enzyme causes surrounding water to be replaced by enzyme
- make substrate more reactive by destabilizing substrate charge
- lowers substrate entropy
Strain & Distortion
- active site conformational change to fit TS
- this substrate distortion increases reactivity
Proximity
- rate increase due to 2 reactants being brought together
- enzymes do this by providing a docking site + micro environment allowing proper substrate orientation for reaction
- contributes to the loss of substrate freedom of movement and loss of entropy
- increase chances of reaction by increasing effective substrate concentration
Km
- Michaelis constant
- higher value means there is lower affinity of the enzyme for the substrate
- need more substrate to achieve 1/2 the Vmax value
