Upload
awen
View
62
Download
0
Tags:
Embed Size (px)
DESCRIPTION
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 - PowerPoint PPT Presentation
Citation preview
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-key model
Induced fit model
Enzymes Accelerate Reaction Rate How?
Enzymes accelerate reaction rate but do 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
Michaelis-Menten Model Accounts for Kinetic Properties of many Enzyme
• 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])
k1
k2
k3
• Michaelie-Menten equationsexplains the kinetic trendseen 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
• 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
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 is 108 to 109 M-1 s-1
So, the upper limit of k3 / KM is 108 to 109 M-1 s-1.
• 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.
Enzyme Inhibition
• 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)
Kinetics of Enzyme Inhibition
• 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 : KI
M = 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]
Kinetics of Enzyme Inhibition
Kinetics of Enzyme Inhibition
• 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]