Enzymes are what?
- proteins with specific globular structures - biological catalysts that increase the rates of reactions
Enzyme function depends on ?
- shape that is complementary to reactant molecules - catalytic interactions between reactant molecules and the more or less flexible binding sites on the enzymes - internal protein motions that extend throughout the molecule.... - they function in crowded gel like conditions
Explain catalytic properties of enzymes!
- increase reaction rates - stabilize the transition state which lowers its energy and thereby lowering the activation energy (energy needed for a sub rate to be transformed into a product) - do not change the eq constant or delta G of a reaction
All catalysts and enzymes only affect the ___ of a reaction
- rate -if a reaction is non spontaneous an enzyme cannot affect the rate.
Enzyme and substrate binding at the active site involves ___ bonds.
- covalent (electrostatic, hydrogen and hydrophobic )
how do enzymes stabilize the transitional state?
- conformational changes made possible via active sites shape and charge disturb that fit a specific substrate....this fit forces the substrate into changing into a shape that favours or resembles the transitional state making the enzyme-substrate complex able to proceed to product.
Enzymes can be regulated to conserve?
energy and raw materials
enzymes work in what kind of conditions?
- non ideal
Equilibrium for reactions use acidity or effective concentration instead of concentration. Activity includes what? Whats its coefficient?
- effect of intermolecular interactions - y a= yc y= activity coefficient - depends on size and charge of the species and the ionic strength of the reaction solution a= activity c= concentration
What are the six types of enzymes classified via reaction catalysis ?
oxidoreductases hydrolases isomerases transferases lyases ligases
- oxidation reduction rection
- transfer of functional groups
-adds water and cleaves a bond..
- removes a group and forms a double bond or adds a group to a double bond ( the reverse reaction)
- bond formation (often the removal of water, the reverse of hydrolysis), requires an energy source (ATP hydrolysis)
velocity of a chemical reaction?
- the change in concentration of substrate (or product) as a function of time
Equation for the rate of enzymes?
v= k[S]n v= initial velocity n= the order of the reaction k= rate constant
What does the order of a reaction depend on?
- how many reactants (or substrates) are involved in the slowest step of the mechanism
First order kinetics?
ex: S---> P n= 1, v= K[S].... if s is doubled so should v - if the rate depends only upon the concentration of one reactant the reaction is first order in the reactant and the reaction overall.
Order ? A+B------->P
v= K[A]m[B]n m- order via A n= order via B = m+n = overal order of reaction - if the rate only depended on A v=K[A]1[B]0....reaction is first order via a, zero via b, 1 over all
Second order kinetics?
if the rate depends on the concentration of both A and B... v=K[A][B]....first order in A, first in B...2 overall
The order of the reaction is determined how?
Pseudo-first order kinetics?
- second order reaction that behaves as if its first order...typically because one reactant (H2O for ex) is present in excess...doubling the excess reactant results in no change in rate....double excess is still excess
Zero order kinetics?
- reaction rate doesn't depend on substrate concentration \ - adding more substrate won't increase the rate L> active sites are filled with substrate....therfeore adding more will not increase the rate...enzyme cannot work fast since its at Vmax
number of molecules colliding in a single step reaction - unimollecular: 1 molecule A-->B - bimollecular: 2 molecules A+B----> C+ D
What is the formula for michaelis-menten ?
Vo= Vmax[S]/ [S]+ Km Vo= initial v Vmax= max velocity Km= michaelis constant (exp found) = [S] at half of vmax!
If the [S] = Km what is the M-M eq?
Vo= Vmax/2 aka km is = to the substrate concentration when Vo = half of Vmax
Michaelis-Menten Plot involves?
Vo vs S L> slight curve to it
- km= 1/2 max V.....its the point on the x axis
Assumptions needed to derive Michealis-Menten Equation?
1. rate of the reverse reaction, E+P---> ES is ignored....this is valid as initial v are measured...when P is very Low....rate equations include concentration of reactamts mot product therefore..... E+S--->ES
2. E+S---> ES= K1
L> ES---> E+S K1-
3. Steady rate assumption: (the rate to formES) = ( the rate to degrade ES);, the substrate enzyme complex ES is in steady state...so ES conc remains constant as a function of time.
- turnover number....... vmax/ total enzyme concentration= # of substrate molecules that an enzyme converts to product per unit time when the enzyme is saturated.
- Kcat/Km = indicator of substrate binding efficiency in low S concentrations... S<
Lower Km = greater affinity of?
- the enzyme for the substrate
L> consider this: two enzyme catalyzed with the same Vmax but different Km values...for this reaction in which the enzyme has a greater affinity for the substrate, it takes less substrate to get to the same rate (half or vmax)....an enzyme with lower affinity for substrate needs a higher substrate concentration to work at the same velocity.
- enyzmes working at their diffusion control limit- their reaction rate is limited only by how fast the substrate can ge to the active site.
International Unit of enzyme activity?
- amount of enzyme to produce 1 micro mol per minute of product
L> specific activity?: #I.U. per mg of protein , a measure of enzyme purification
(kat)= 1 mole of substrate converted to product per second.
- determination of Km and Vmax
L> accurate values of Vmax and Km are mch easier to determine using a straight line plot...vmax is actually met here not in M-M plot
-compring of L-B inhibited vs uninhibited can indicate the enzyme inhibition mech
1/Vo= (Km/Vmax)(1/[S]) + 1/vmax
y = m x b
m= slope.....= km/vmax
b= y-intercept= 1/vmax
- rxn involving two or more substrates.
- two types: sequential and double displacement reactions
- it cannot proceed until all substrates have bound in the enzymes active site...ordered or random.
- ordered: first substrate must bind before the second binds for the reaction to occur....in random any can bind at at order.
Double displacement reactions?
- ping pong mech
- the first product is released before the second substrate binds....the enzyme is altered by the first phase of the rxn .....the enzyme is then restored to its original form during the second phase in which the second substrate is converted to product.
- first sub binds and releases the first product before the second sub binds
- activity of an enzyme can be inhibited by these molecules which reduce its activity.
- inibition can be reversible or irreversible
- inhibitory effect of a compound can be counteracted by increasing substrate levels or removing the inhibitor compound while the enzyme remains intact
Types of reversible inhibition?
- competitive and noncompetitive
- inhibitor binds to the free enzyme and competes with the substrate for occupation of the active site.
- if the inhibitor binds to both the free enzume and the enzyme substrate complex
- inhibitor only binds to the ES
- inhibitor binding permanetly impairs the enzyme via covlent reaction that chem modifies the enzyme.
Allosteric enzymes cannot be expressed by which plot?
- many macromolecules crowd impeding molecular movement within a cell.
Transition State vs Intermediate?
- active site stabalizes in the t state and has a greater affinity for the trasition state..vs for substrates or products.
- intermediate: A species that exists for afinite length of time...then transformed into product. (carbonocation, carbanion and free radicals)
- Proximity and strain effects; electrostatic effects
L> the more tightly and efficiently that an active site can bind substrate while its in its transition state the faster the reaction will be
-you want a weak electrostatic effect....hydration shells will increase distance between charge centers and reduce electrostatic attraction.
- Acid-base catalysis
- amino acids with side chains acting as either...depend ont eir pKa and environment.
- covalent catalysis
- nucleophilic amino acids like ser form weak covalent bonds to facilitate the reaction
Quantumn tunneling and enzyme catalysis?
-travelling through an energy barrier rather than over it...helps the very high rates enzymes can achieve.
Describe catalytic aa's side chains?
- polar or charged side chians
Dyads or triads?
groups of 2 or three aa that are positioned perceisely for catalytic effects...such as polarizing a specific atom or gorup, or chaning pKa of anearby functional gorup...
Noncatalytic aa's functions?
- function in substrate orientation and transition state stabilization..
- non protein components that add chemical functionality to enzymes and this increase the kinds of reactions that enzymes can catalyze
L> transition metals
effects of temp and pH on enzyme-catalyzed reactions!
- Temp and pH changes affect tertiary structures of proteins...which must be very specific for enzymes to function at their vmax...
- higher temps generally increase rates but denaturation can occur if too high
- maintains an ordered state, conserves energy and helps the cell to respond to environmental changes. usually xontroled by covalent modification or allosteric regulation
Enzyme reg types?
1. Genetic control
2. Covalent modification
3. allosteric regulation
- enzyme induction and repression
- enzymes are synthesized when needed
- enzyme synthesis is inhibited
Enzyme reg mech:
L>phosphorylation and zymogens
- convert an enzyme between its active and inactive form
- proenzymes.... converted into active enzymes by an irreversible cleavage of one or more peptide bonds.
-ligands bind to allosteric site and trigger conformational changes
- importance of the flex of proteins.
- heterotrophic: LIGAND IS NOT THE SUB
Concerted (symmetry) model for allosteric
-activators bind to and stabilize the t conformation....conformation of all subunits are believed to change adter the first effector binds.
sequentual model of allosteric effects
- upond binding the effector , subunits change sequentially
Positive cooperativity ?
firs ligand increases subsequent ligand binding
- first ligand reduces the enzymes affinity for similar ligands.
- enzymes, substrates and reg molecules are located in sep compartments so that opposing pathways are physically separated or located close.
Compartmentation solves?? (4)
1. divide and control: sep of enzymes into different regions or compartments to use resources efficiently....give cell more control..via controlling transport of substrates, products, effectors etc across organelle membranes (ex: mitochondria membranes)
2. Diffusion barriers: microenvironments that conentrate enzymes and or substrates and so reduce diffusion barriers. Enzymes located close to each other can increase the efficiency of coupled rxns and or of entire pathways (such as electron tc) aka metabolic channeling
3. specialized reaction conditions
4. damage control : segregation of molecules that may be within a toxic cell.