Loading in 5 sec....

General Features of EnzymesPowerPoint Presentation

General Features of Enzymes

- By
**awen** - Follow User

- 144 Views
- Uploaded on

Download Presentation
## PowerPoint Slideshow about ' General Features of Enzymes' - awen

**An Image/Link below is provided (as is) to download presentation**

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.

- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript

General Features of Enzymes

- Most biological reactions are catalyzed by enzymes
- Most enzymes are proteins
- Highly specific (in reaction & reactants)
- Involvement of cofactor or coenzyme in some enzymes
(prosthetic groups, holoenzyme, apoenzyme)

- Activity regulated through
- Feedback inhibition
- Regulatory proteins (e.g. calmodulin)
- Covalent modification (e.g. phosphorylation)
- Precursor to mature form transition
(proteolytic activation)

How Enzymes Work

- Substrate binding is the first step of enzymatic catalysis
- Substrate
- Active site
- Binds substrate (by multiple weak interactions)
- A 3-dimensional entity complementary to substrate
- Contains catalytic residues
- Size and location: Small; located at clefts or crevices
- Source of binding specificity

- Enzyme-substrate interaction:
- Lock-and-keymodel
- Induced fit model

Enzymes Accelerate Reaction Rate

How?

- Enzymes accelerate reaction ratebutdo not alter equilibrium!
- Rate of reaction= (Ae-G‡/RT)[S]
- Accelerate reaction rate by stabilizing transition states (G‡)
- Essence of catalysis: specific binding of the transition state

k1

k2

Michaelis-Menten ModelAccounts for Kinetic Properties of many Enzymek3

- Kinetic properties of many enzymes (V vs. [S] plot)
- Michaelis-Menten Model
E + S ES E + P

- Purpose: using the model to derive an expression relating
rate of reaction to [E] and [S] and k1, k2, and k3

- Assumption #1: no product reverts to initial substrate (initial state)
- Assumption #2: steady state ([ES] is constant)
- k1[E][S]=k2[ES]+k3[ES], so [ES] = [E][S]/KM ; KM =(k2+k3)/k1
- [E] = [ET] - [ES]; [S] = [ST] - [ES] - [P]
- work under the following condition: [ET] << [ST] ; and at initial time, so [P] is negligible, and so [S] = [ST] [ES] = [ET] [S]/(KM + [S])
so, V = k3 [ES] = k3[ET] [S]/(KM + [S]) = Vmax [S]/(KM + [S])

- Purpose: using the model to derive an expression relating

- Michaelie-Menten equations
explains the kinetic trend

seen for many enzymes

V = Vmax [S]/(KM + [S]):

- When [S] << KM, V = Vmax [S]/KM ,
V is directly proportional to [S]

- When [S] >> KM , V = Vmax ,
rate is maximal, independent of [S]

- When [S] = KM, V = (1/2) Vmax,
so, KM = [S] when V is 1/2 Vmax

- When [S] << KM, V = Vmax [S]/KM ,

- Determine KM and Vmax
- Experimental Procedure
- Set up several reactions with fixed [ET] but increasing [ST]
- Experimentally determine V at various [ST] (simplified as [S];
V is initial velocity so [P] is negligible)

- Data Analysis
- Using Michaelis-Menten Equation:
V = Vmax [S]/(KM + [S])

- Plot V vs. [S]; computer curve fitting to find KM and Vmax

- Lineweaver-Burk Plot
1/V = 1/Vmax + (KM/Vmax) 1/[S]

- Plot 1/V vs. 1/[S]
- Y intercept = 1/Vmax; X intercept = -1/KM

- Using Michaelis-Menten Equation:

- Experimental Procedure

Kinetic Perfection in Enzymatic Catalysis

- For Enzymes that Obey Michaelis-Menten Model
- When all enzyme molecules are saturated with substrate
- V = Vmax = k3 [ET], rate constant is k3 (= kcat)

- When [S] << KM and so most of the active sites are unoccupied
- V = k3 [ES]= k3 [E][S]/KM
as [S] << KM, so [E] [ET], so V = k3 [ET][S]/KM = (k3/KM)[ET][S]

so V depends on k3 / KM: k3 / KM= k3 k1 / (k2 + k3) < k1

k1 cannot be faster than diffusion controlled encounter of

an enzyme and its substrate, which is108 to 109 M-1 s-1

So, the upper limit of k3 / KM is 108 to 109 M-1 s-1.

- V = k3 [ES]= k3 [E][S]/KM

- When all enzyme molecules are saturated with substrate
- For Enzymes that Do not Obey Michaelis-Menten Model
- When all E are saturated with S, rate depends on k cat; kcat k3
- When not all E are saturated with S, rate depends on k cat / KM

- Some enzymes having k3/KM of 108 - 109 M-1 s-1 reached kinetic perfection! Their catalytic velocity is limited by the rate at which they encounter substrate in the solution.

- Irreversible Inhibition
- Inhibitor destroys a functional group on the enzyme
- Or inhibitor binds to the enzyme very tightly (covalently or noncovalently) dissociates very slowly from enzyme

- Reversible Inhibition

- Reversible Inhibition
- Inhibitor binds and dissociate rapidly from the enzyme
- Competitive inhibitor
- Inhibitor binds at active site; compete for binding with substrate; exist as either ES or EI; no ESI
- Inhibitor structure resembles that of substrate
- Overcome competitive inhibition by increasing [S]

- Noncompetitive inhibitor
- Inhibitor binds at a site other than active site
- Binding of noncompetitive inhibitor decreases turnover number (reduces k3)

- Assume the enzyme exhibits Michaelis-Menten Kinetics
- Set up enzymatic reactions with fixed [ET] but increasing [ST]
- One set without inhibitor and another set with inhibitor
- Plot 1/V vs. 1/[S] (Lineweaver-Burk Plot)

- Competitive Inhibition
- The two lines on the plot have the same Y intercept (Same V max)
- KM and KIM are different : KIM = KM (1 + [I]/KI)
KI = [E][I]/[EI] (for E + I EI)

- 1/V = 1/Vmax + KM/Vmax (1 + [I]/KI) (1/[S])
- KM and KIM can be determined from the Lineweaver-Burk plot
- KM’ = KM (1 + [I]/KI) allows the determination of KI
- Inhibition can be overcome
by increasing [S]

- Noncompetitive Inhibition
- Same KM in the presence and absence of Inhibitor
- Smaller V max in the presence of Inhibitor
- VI max = V max /(1 + [I]/KI)
- VI max and V max can be determined from the Lineweaver-Burk plot
- VI max = V max /(1 + [I]/KI)
allows the determination of KI

- Cannot be overcome
by increasing [S]

Download Presentation

Connecting to Server..