L10-wk5-Enzyme kinetics

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L10-wk5-Enzyme kinetics Door Mind Map: L10-wk5-Enzyme kinetics

1. Enzyme substrate complexes (ES)

1.1. formation of ES complex=1st step in enzymatic catalysis

1.2. enzymes bring substrates together in an ES complex

1.2.1. favourable orientation for formation of transition state

1.3. substrates bind to a specific region on the enzyme

1.3.1. active site

1.4. enzymes are highly selective in substrates that they bind

1.5. catalytic specificity depends on specificity of binding

1.6. activity of many enzymes regulated at this stage

1.7. Evidence for ES complexes

1.7.1. the fact that at a constant concentration of enzyme, rate of rxn increases with increasing substrate conc. until a maximal velocity is reached

1.7.1.1. Maximal velocity=Vmax

1.7.1.2. uncatalyzed rxn doesnt show saturation effect

1.7.1.3. fact that enzyme catalyzed rxn has Vmax

1.7.1.3.1. suggests the formation of a discrete ES complex

1.7.1.4. Vmax

1.7.1.4.1. rate/velocity at which all enzyme active sites are filled

1.7.1.5. at a high substrate conc.

1.7.1.5.1. all catalytic sites on the enzyme are filled

1.7.1.5.2. saturation

1.7.1.5.3. rxn rate cannot increase

1.7.1.6. ability to saturate an enzyme with substrate=evidence for existence of ES complex

1.7.2. Structural data from X-ray crystallography

1.7.2.1. provides high res images of substrates and substrate analogs bound to ASs of many enzymes

1.7.2.2. enzyme cytochrome P450 illustrated bound to its substrate camphor

1.7.2.3. in active site

1.7.2.3.1. substrate is surrounded by residues from the enzyme

1.7.2.3.2. heme grp cofactor present

1.7.2.4. carried out at low temps to slow rxn down

1.7.3. spectroscopic data

1.7.3.1. spectroscopic characteristics of many enzymes and substrates change on the formation of the ES complex

1.7.3.2. changes particularly striking if the enzyme contains a coloured prosthetic grp

1.7.3.3. Absorption and fluorescence of proteins+cofactors change when mixed together

1.7.3.4. example

1.7.3.4.1. Spec of tryptophan synthetase

2. Active site

2.1. =region of an enzyme that binds the substrates

2.1.1. and cofactors if there are any

2.2. interaction of the enzyme and substrate at the active site promotes the formation of the transition state

2.3. contains catalytic grps

2.3.1. =residues that directly participate in making and breaking bonds

2.4. active site generalizations

2.4.1. AS=a 3D cleft/crevice

2.4.1.1. formed by groups that come from different parts of the amino acid sequence

2.4.1.2. amino acid residues are far apart in the amino acid sequence

2.4.1.2.1. interact more strongly than adjacent residues

2.4.1.2.2. adjacent residues may exhibit steric hindrance

2.4.1.3. example

2.4.1.3.1. lysozyme

2.4.2. active site takes up a small part of the total volume of the enzyme

2.4.2.1. most AA residues of the enzyme are not involved in the AS

2.4.2.2. extra amino acids serves as the scaffold that creates the 3D active site

2.4.2.3. remaining AAs make up the regulatory sites

2.4.2.3.1. sites of interaction with other proteins

2.4.2.3.2. channels to bring the substrates to the active sites

2.4.3. active sites are unique microenvironments

2.4.3.1. ASs are shaped like a cleft/crevice

2.4.3.1.1. substrates bind

2.4.3.2. water usually excluded(unless its a reactant)

2.4.3.2.1. non polar microenvironment

2.4.3.3. non polar microenvironment

2.4.3.3.1. enhances the binding of substrates and catalysis

2.4.3.3.2. also contains some polar residues

2.4.4. Substrates are bound to enzymes by multiple weak interactions

2.4.4.1. noncovalent interactions

2.4.4.1.1. weaker than covalent bonds

2.4.4.1.2. Equilibrium constants of ES complexes

2.4.4.1.3. Free energies of ES complexes

2.4.4.1.4. weak irreversible interactions

2.4.5. Specificity of binding depends on the precise defined arrangement of atoms in the AS

2.4.5.1. Enzyme and substrates interact by short range forces (require close contact)

2.4.5.1.1. substrate must have complementary shape to fit into AS

2.4.5.1.2. atoms in substrate align with atoms in enzyme

2.4.5.2. Lock and key model 1890

2.4.5.2.1. Emil Fischer

2.4.5.2.2. active site of the unbound enzyme is complementary in shape to the substrate

2.4.5.2.3. substrate + active site are initially complementary in shape

2.4.5.3. Induce fit model 1958

2.4.5.3.1. Daniel E. Koshland Jr.

2.4.5.3.2. substrates and enzymes not rigid structures

2.4.5.3.3. shapes of the ASs can be modified by binding of the substrate

2.4.5.3.4. enzyme changes shape only on substrate binding

3. Binding energy

3.1. free energy is released by the formation of a large number of weak interactions between the enzyme and substrate (EScomplexes)

3.2. binding energy

3.2.1. free energy released when a substrate binds to its complementary enzyme

3.3. only the correct substrate can take part in all enzyme interactions

3.3.1. maximize binding energy

3.4. full set of interactions are only formed when the substrate is converted > transition state

3.4.1. maximal binding energy is released when enzyme facilitates formation of the TS

3.4.2. most stable interactions take place between enzyme and transition state

3.5. transition state is too unstable to exist for long

3.5.1. collapses>substrate/product

3.5.2. which one it forms depends on free energy difference between substrate and product =∆G

4. Enzyme kinetics

4.1. kinetics

4.1.1. the study of the rates of chemical rxns

4.2. enzyme kinetics

4.2.1. the study of the rates of enzyme catalyzed rxns

4.3. 1st order rxns

4.3.1. reactions that are directly proportional to the reactant concentration

4.3.2. uni-molecular

4.3.3. consider eqn

4.3.3.1. rate(V)

4.3.3.1.1. quantity of A(substrate/reactant) that disappears in a specific unit of time

4.3.3.1.2. quantity of P(product) that appears in a specified unit of time

4.3.3.2. if we follow rxn by seeing what happens to A

4.3.3.2.1. if A is yellow and P is colourless

4.3.3.2.2. we measure the decrease in [A] by measuring decrease in intensity of colour

4.3.3.2.3. rate of rxn is dependent on

4.4. 2nd order rxns

4.4.1. bimolecular

4.4.2. many impt. biochemical rxns include 2 reactants

4.4.3. rxn equation

4.4.3.1. 2nd order rate constants k

4.4.3.1.1. units

4.4.3.2. can also be V=k[A]^2

4.5. pseudo first order rxns

4.5.1. second order rxns can appear to be first order rxns

4.5.2. if B is present in excess and A is present at low cons

4.5.2.1. [B]>>[A]

4.5.2.2. rxn rate will be 1st order with respect to A and will not appear to depend on conc. of B

4.5.3. if A is present in excess and B is present at low cons.

4.5.3.1. [A]>>[B]

4.5.3.2. rxn rate will be first order with respect to B and will not appear to depend on conc. of A

4.6. zero order rxn

4.6.1. the rate is independent of reactant concentrations

4.6.2. enzyme catalyzed rxns can approximate 0 order rxns under some circumstances

4.7. The steady state assumption

4.7.1. simplest way to investigate rxn rate

4.7.1.1. follow the increase in rxn product as a function of time

4.7.1.2. amount of product formed increases with time

4.7.1.2.1. reaches a time when there is no net change in conc. of S/P

4.7.1.2.2. initial velocity (V0)

4.7.2. Determining the relation between intitial velocity and substrate conc

4.7.2.1. considering only > rxn rate of catalysis Vo=

4.7.2.1.1. number of moles of product formed/s when the rxn is just beginning (t~0)

4.7.2.1.2. initial velocity

4.7.2.2. assume amount of enzyme present is constant

4.7.2.3. plot Vo against [S]

4.7.2.3.1. many enzymes yield similar results

4.7.2.3.2. results

4.7.3. Michaelis Menten Model

4.7.3.1. 1913

4.7.3.1.1. Leonor Michaelis and Maud Menten proposed a simple model to account for kinetic characteristics of enzymes

4.7.3.2. critical feature

4.7.3.2.1. the fact that a maximum rxn velocity is reached with saturated substrate and constant enzyme concentration

4.7.3.2.2. implies that an ES complex must be present as an intermediate in enzyme catalysis

4.7.3.3. Michaelis menten kinetics

4.7.3.3.1. plot of rxn velocity(V0) against substrate conc[S]

4.7.3.3.2. enzyme obeys Michaelis Menten kinetics

4.7.3.3.3. maximal velocity Vmax is approached asymptomatically

4.7.3.3.4. Vmax

4.7.3.3.5. KM

4.7.3.3.6. rate is initially proportional to [S]

4.7.3.3.7. proportionality lost and curve levels off as Vmax(saturation) is reached

4.7.3.4. model proposed

4.7.3.4.1. rxn eqn

4.7.3.4.2. simplified by considering the rate of rxn close to 0=V0

4.7.3.4.3. catalytic rate=the product concentration of the ES complex and k+2

4.7.3.4.4. express [ES] in terms of known quantities

4.7.4. The steady state assumption

4.7.4.1. proposed by George Briggs and John Haldane 1924

4.7.4.2. in steady state

4.7.4.2.1. concentrations of intermediates [ES]

4.7.4.2.2. reached when the rate of formation of ES=rate of breadown of the ES

4.7.4.2.3. rearrange eqn to get

4.7.4.2.4. get eqn [ES]=[E][S]/KM

4.7.4.2.5. sub eqn [E]= into [ES] eqn

4.7.4.2.6. sub [ES] final eqn into V0 equation

4.7.4.2.7. Vmax(maximal rate)

4.7.4.2.8. Substitute eqn back into V0

4.7.4.2.9. Vmax and Km are measurable constants for a particular enzyme rxn

4.8. Significance of Vmax

4.8.1. maximal rate Vmax

4.8.1.1. reveals the turnover number of an enzyme

4.8.1.2. turnover number

4.8.1.2.1. the number of substrate molecules converted into a product by an enzyme molecule in a unit time when the enzyme is fully saturated with substrate

4.8.1.2.2. =rate constant k+2

4.8.1.3. reveals the turnover number of an enzyme if the concentration of the active sites [E]T is known

4.8.1.3.1. [ES]=[E]T

4.8.1.3.2. [E]T

4.9. Significance of KM

4.9.1. very low substrate concentration

4.9.1.1. [S]<<KM

4.9.1.2. V0=(Vmax/Km)[S]

4.9.1.3. rxn is first order with the rate directly proportional to the substrate conc.

4.9.1.3.1. both Vmax and Km are constants

4.9.2. high substrate concentration

4.9.2.1. [S]>>KM

4.9.2.2. V0=Vmax

4.9.2.2.1. rate is maximal

4.9.2.3. rxn is 0 order

4.9.2.3.1. independent of substrate concentration

4.9.3. When [S]=KM

4.9.3.1. V0=Vmax/2

4.9.3.2. KM

4.9.3.2.1. =the substrate concentration at which the reaction rate is half its maximal value

4.9.4. KM for any enzyme depends on

4.9.4.1. pH

4.9.4.2. Temp

4.9.4.3. ionic strength

4.9.5. KM

4.9.5.1. conc. of substrate required for rxn velocity 2 be half its maximal value

4.9.5.2. Vmax/2

4.9.5.3. When S=KM

4.9.5.3.1. V=Vmax/2

4.9.6. if k+2<<k-1

4.9.6.1. KM=dissociation constant of the ES complex

4.9.6.2. little product is formed

4.9.6.3. take k+2 out

4.9.6.4. under these conditions KM tell us the enzyme-substrate complex affinity

4.9.6.4.1. only when k-1>>k+2

4.9.6.5. KM is a measure of the strength of the ES complex

4.9.6.5.1. small KM=strong binding

4.9.6.5.2. large KM=weak binding

4.10. Catalytic power/efficiency

4.10.1. when the substrate conc. >> KM

4.10.1.1. rate of catalysis=Vmax

4.10.1.2. Vmax is a function of kcat(turnover number)

4.10.2. enzyme's turnover=catalytic power

4.10.3. catalytic power

4.10.3.1. the max # of substrate molecules converted into product by the enzyme molecule/unit time

4.10.4. when E is fully saturated=kinetic constant(kcat/k+2)

4.10.4.1. kcat can be calculated from Vmax

4.10.4.2. Vmax=kcat[E]T

4.10.5. sub Vmax eqn into V0 equation

4.10.5.1. Vmax

4.10.5.2. V0

4.10.5.3. = new eqn

4.10.5.4. kcat/KM

4.10.5.4.1. measure of catalytic power

4.10.5.4.2. takes into account

4.10.5.4.3. by using kcat/Km values

4.10.6. rate will increase with [E]T and [S]

4.10.7. diffusion limit

4.10.7.1. maximum value of kcat/Km

4.10.8. V0=velocity of the rxn is dependent on

4.10.8.1. # of active sites

4.10.8.2. substrate conc

4.10.8.3. diffusion limit

4.10.8.3.1. # of active sites & substrate conc.

4.10.8.4. depends on collision of substrates and active sites

4.11. Perfect enzyme

4.11.1. is limited by diffusion

4.11.2. ultimate limit on the value of kcat/Km is set by k1

4.11.2.1. k+1=the rate of formation of the ES complex

4.11.2.2. rate cannot be faster than the diffusion-controlled encounter of an enzyme and its substrate

4.11.3. Diffusion limits value of k1

4.11.3.1. cannot be higher than

4.11.3.1.1. 10^8 - 10^9 s^-1M^-1

4.11.3.2. limits value of kcat/KM

4.11.3.2.1. can be in range of 10^6 - 10^9 s^-1M^-1

4.11.3.2.2. upper limit

4.11.3.3. k1

4.11.3.3.1. a measure of how often an enzyme collides with its substrate

4.11.4. kineticly perfect enzymes

4.11.4.1. those that have kcat/KM ratios at the upper limits

4.11.4.2. their catalytic velocity is restricted only by the rate at which they encounter a substrate in solution

4.11.4.2.1. diffusion limited

4.11.4.3. e.g. acetylcholinesterase and triose phosphate isomerase

4.11.4.4. don't slow enzyme rate down

4.11.4.4.1. lower k2/KM=slow enzyme rate down

4.11.5. further gain in catalytic rate

4.11.5.1. decrease the time the substrate takes to diffuse into the enzymes environmennt

4.11.5.2. Circe effects

4.11.5.2.1. attractive electrostatic forces on the enzyme that entice/lure the substrate to the active site

4.11.5.2.2. the utilization of attractive electrostatic forces to lure a substrate into a site in which it undergoes a transformation of structure

4.11.5.2.3. derived from greek methology

4.11.5.3. diffusion can also be partly overcome by confining substrates and products in a limited volume

4.11.5.3.1. e.g. a multienzyme complex

4.11.5.3.2. products are channeled from one enzyme to the nezt

4.11.5.3.3. products rapidly available for the next enzyme

5. Multiple substrate rxns

5.1. most rxns in biological systems start with 2 substrates and yield 2 products

5.1.1. represented by the bisubstrate rxn

5.2. many such rxns

5.2.1. transfer a functional grp from one substrate to another

5.2.1.1. phosphoryl

5.2.1.2. ammonium grp

5.2.2. oxidation-reduction

5.2.2.1. transfer e-s between substrates

5.3. divided into 2 classes

5.3.1. sequential rxns

5.3.1.1. all substrates must bind to the enzyme b4 any product is released

5.3.1.2. ternary complex forms

5.3.1.2.1. consists of the enzyme + both substrates

5.3.1.3. two types of sequential mehanisms

5.3.1.3.1. ordered

5.3.1.3.2. random

5.3.2. double displacement rxns

5.3.2.1. ping pong rxns

5.3.2.1.1. in Cleland notation substrates appear to bounce on and off enzyme

5.3.2.1.2. like a ping pong bounces on a table

5.3.2.2. one or more products are released b4 all substrates bind the enzyme

5.3.2.3. defining feature

5.3.2.3.1. existence of a substituted enzyme intermediate

5.3.2.3.2. substituted enzyme intermediate

5.3.2.4. e.g. rxns that shuttle amino grps between AAs

5.3.2.4.1. alpha ketoacids

5.3.3. Cleland notation

5.3.3.1. used to show an enzyme mechanism as a sequence of events on a timeline

6. Limitations of Michaelis Menten

6.1. M-M kinetics is a simple model of enzymology

6.2. cannot account for the kinetic properties of many enzymes

6.3. there are many enzymes that don't follow M-M kinetics

6.3.1. impt. grp that does not obey M-M kinetics are allosteric enzymes

6.3.1.1. consist of multiple subunits and multiple Active sites

6.3.1.2. display sigmoidal plots of rxn velocity (V0) vs. substrate concentrations [S}

6.3.1.2.1. not hyperbolic like M-M enzymatics

6.3.1.2.2. bcuz they often exhibit cooperation

6.3.1.3. binding of substrate to one active site can alter properties of other active sites in the same enzyme molecule

6.3.1.4. activity can be altered by regulatory molecules that are reversibly bound to specific sites other than the catalytic sites

6.3.1.4.1. catalytic properties can be adjusted to meet the immediate needs of a cell

7. Summary

7.1. enzymes form complexes with their substrates

7.2. Substrates bind to active sites of enzymes

7.2.1. influenced by tertiary structure of protein

7.3. KM=

7.3.1. the concentration of substrate needed 4 the rxn to be 1/2 the maximum value

7.3.2. Vmax/2

7.4. Vmax=

7.4.1. maximum rate of rxn when the enzyme is fully saturated with substrate