Cover Slide

MCMP 304 September 26, 2011

MCMP 304 9/26/11 1

ES Complex Explains Saturation Kinetics

The approach to zero order kinetics occurs because catalyzed reactions involve an intermediate: complexes with the catalyst (E): E + S ES E[TS*] EP E + P

When nearly all of the enzyme is in one of these complexes, adding more S does not significantly increase the amount of ES This is because there is little or no free E to complex with the added S The amount of E complexes is maxed out and is limited by total amount of E, so more S has no impact At that point the enzyme is said to be saturated
Topic 10.7

MCMP 304 9/26/11 2

The Michaelis-Menton Model of Enzyme Kinetics

E+S 1 2 ES/E[TS*]/EP

3
4

E+P

(Equation 10.16)

This is the Michaelis-Menton Vmax [S] model. (Equation 10.19) v= [S] + KM [S] is the initial substrate concentration and v is the initial velocity of the reaction. Where: k2 + k3 Vmax = k3 [E]Total and KM = k1 (k1, k2, and k3 are the rate constants for reactions 1, 2, and 3) and assuming that: [E]Total is constant, v1 + v4 = v2 + v3 ; and vF >> vR
Topic 10.7

MCMP 304 9/26/11 3

The Assumptions of the Michaelis-Menton Kinetic Model for Enzymes

See Closer Look 10.5 box on page 399 for model derivation

The Steady State Assumption: v1 + v4 = v2 + v3 Rate of E complex formation = Rate of E complex breakdown
[E-complex] is constant, hence the enzyme is in a steady state This is almost always true and is not of general concern experimentally.

vF >> vR The reverse reaction is negligible. This is almost never true in cells where almost all reactions are close to equilibrium. The only time this is usually true is in experimental solutions that initially lack any product when substrate and enzyme are first mixed together, allowing observation of the initial velocity. Note that the v in the Michaelis-Menton equation is an initial velocity, not an instantaneous velocity, solely because of this assumption. MCMP 304 9/26/11 4 Topic 10.7

As vR approaches vF : the reverse reaction rate becomes so great that it can no longer be
ignored and the Michalis-Menton equation is no longer valid. However, there are modified forms of the model that take product inhibition into account. the observed velocity of the reaction (the net reaction in the forward direction) slows below that predicted by the Fig 10.47 model. The same result is seen if ETotal is not constant due to loss/decay of enzyme activity due to instability of the enzyme during the assay but the cause of this is different. MCMP 304 9/26/11 5 Topic 10.7

Violation of the vF >> vR assumption leads to product inhibition

The Value of the Michaelis-Menton Kinetic Model for Enzymes

Michaelis-Menton kinetics are observed only under experimental conditions. However, their kinetic model parameters (KM and Vmax) provide information that is useful beyond the experimental conditions used.
The kinetic parameters must be determined empirically under conditions where the assumptions of the model are valid. KM and Vmax cannot be observed directly, but can be deduced from fitting the model equation to the observed experimental data: the initial reaction velocity (v) at a specific initial substrate concentration ([S]). Valid conditions must be used to measure the initial velocity, as shown by a validation experiment that demonstrates that at the same [E] and at the lowest and highest [S] used, the velocity is constant during the measurement of the initial velocities.
Topic 10.7

MCMP 304 9/26/11 6

Kinetic Parameters
4.5

Vmax
4 3.5 3 2.5 2 1.5

v0

Vmax

Similar to Fig 10.48

1 0.5 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

KM

[S] Topic 10.7

MCMP 304 9/26/11 7

KM
KM =

k2 + k3 k1

KM is the enzyme analog of an affinity constant (dissociation equilibrium constant) and always has units of concentration.

When [S0] = KM , then v0 =

Vmax KM KM + KM = Vmax

which is also called the half-maximal velocity

Topic 10.7

MCMP 304 9/26/11 8

KM Indicates the Dependence on S0 for Approach to Vmax

4.5 4 3.5 3 2.5

Vmax= 4.2

Km= 0.1 Km= 0.5

Vmax= 2.1

v0

2 1.5 1 0.5 0 0 0.5 1 1.5 2 2.5 3

Km= 2.5

3.5

4.5

[S0]

Topic 10.7

MCMP 304 9/26/11 9

Vmax (=k3 [E]Total) is proportional to the amount of catalytic activity (active enzyme) present. So it is a good measure of enzyme activity.

Vmax

Specific experimental conditions are needed to determine Vmax: steady state, negligible reverse-reaction, saturating [S0], stable enzyme.
Vmax = Enzyme Activity (rate units) for a sample of enzyme Vmax/[protein] = Specific Activity of the enzyme (indicates purity) kcat (= k3) or turnover number - a constant for a given substrate and enzyme. Units of reactions per second. It indicates catalytic efficacy of an enzyme and is suitable for comparing different enzymes or mutants of the same enzyme. MCMP 304 9/26/11 10 Topic 10.7

Different Vmax with the Same KM

4.5 4 3.5 3 2.5

Vmax= 4.2

v0

2 1.5 1 0.5 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Vmax= 2.1

Vmax= 1.2

Km=0.5

[S0]

Topic 10.7

MCMP 304 9/26/11 11

Experimentally determine, using valid conditions, the initial forward reaction velocity (v0) for a fixed amount of enzyme at each of several different initial substrate concentrations.

Determining KM and Vmax

The observed data (result set) consists of many data pairs of initial velocities at each of several substrate concentrations ([S0],v0). Use non-linear regression to fit the data pairs to the Michaelis-Menton model and produce maximum likelihood estimates for the KM and Vmax.
v0 = Vmax [S] [S] + KM ax y= x+b
Non-linear
Topic 10.7

y = a +bx +cx2
Polynomial MCMP 304 9/26/11 12

Analogy to Receptors: KM Can Be Thought of as the Enzyme Analog of an Affinity Constant

Receptor Binding
1
R+L 2 RL

Enzyme-Substrate Binding
1 E+S 2 3 ES(EP) E + P

k2 + k3 KM = k1 This analogy is not perfect. It is an approximate concept

k2 Kd = k1

because k3 can never be neglected as zero for an enzyme

and thus some substrate is always lost from ES complex as product and so a true binding equilibrium never happens with

enzymes. k3 is never zero for an enzyme because enzymes

really bind [TS*] not S or P, in contrast to receptors.
Topic 10.7

MCMP 304 9/26/11 13

The ratio kcat/KM

At [S] << KM: Vmax [S0] v0 = [S ] + K 0 M becomes:

Vmax [S0] kcat [Etotal] [S0] v0 = KM KM

This ratio of kcat/KM is the slope of the other asymptote that characterizes the hyperbola (other than the Vmax line). When viewed as a rate law, this ratio is a second order rate constant (v=k [E][S]). Second order reactions cannot occur faster than diffusion. So this ratio has an upper limit at physiological temperatures of 109. For most enzymes it is about 107. This tends to limit variation in kcat and KM so that if one is larger, the other must be smaller. MCMP 304 9/26/11 14 Topic 10.7

Linear Transformations
The equation for the Michaelis-Menton model (a nonlinear model) can be rearranged in several ways to a form representing a straight line (y = ax+b). The Lineweaver-Burk plot is one of three different linear transformations of the Michaelis-Menton equation. (The other two are Eadie-Hofstee and HanesWoofe.) The Lineweaver-Burk plot is the most popular. Linear transformations were invented to estimate enzyme kinetic parameters using linear regression (fitting empirical data to a polynomial function). The intercepts of the Lineweaver-Burk plot give the reciprocals of the kinetic constants.
Topic 10.7

MCMP 304 9/26/11 15

The Lineweaver-Burk Plot

1 v0 = y = KM 1 1 Vmax [S0] + Vmax a
(slope)

(y-intercept)

Plot 1/v vs 1/S0

Hence the nickname: Double reciprocal plot

Figure 10.49
Topic 10.7

MCMP 304 9/26/11 16

All Linear Transformations Can Be Highly Inaccurate For Calculating Kinetic Constants from Experimental Data
Linear transformation of the Michaelis-Menton equation alters the weight that should be placed on each point when doing the linear regression to fit the straight line and find the slope and intercept, and from them the Km and VMax If a linear transformation is used to calculate kinetic constants, the fitting of the transformed data must be done using weighted linear regression, with the error on the data properly propagated through the transformation into the weights. If this is not done, the fitted line (defined by its slope and intercept) is subject to higher error than if the data had been properly weighted during the linear regression. For instance, if unweighted, the the Lineweaver-Burk plot overweights points where S0 < Km MCMP 304 9/26/11 17 Topic 10.7

Non-linear Regression is Always the Best Way to Calculate Kinetic Constants from Experimental Kinetic Data
Using linear regression with transforms of the MichaelisMention equation without properly weighting the data results in larger errors in the kinetic constants estimated from the data.

Fitting of data to transformed equations is commonly NOT done with proper weighting because most linear regression programs (e.g., Excel, programmable calculators) do not provide a simple way to do this.
Non-linear regression is the proper way to fit (v0,S0) data to the non-linear Michaelis-Menton kinetic model, since it does not transform the equation at all.
Topic 10.7

MCMP 304 9/26/11 18

Linear Transformations Are Useful for Visualization of the Kinetic Parameters

The Lineweaver-Burk plot can help visualize whether two sets of data have different or same Vmax or different or same KM (both parameters should be found by non-linear regression). For the Lineweaver-Burk plot:
X-intercept = -1/KM Y-intercept= 1/Vmax Slope = KM/Vmax

Comparison of different Vmax at the same KM:

These double reciprocal plots will have the same X-intercept but different slopes (and hence different Y intercepts)

Comparison of same Vmax at the different KM:

These double reciprocal plots will have the same Y-intercept but different slopes (and hence different X intercepts)
Topic 10.7

MCMP 304 9/26/11 19

Comparison of Different KM At the Same Vmax

3

2.5

1.5

Vmax= 4.2 1/Vmax= 0.24 for all three

1/v 0

KM= 0.1 -1/KM= -10

-11 -10 -9 -8 -7 -6 -5

KM= 0.5 -1/KM= -2

-4 -3 -2 -1

0.5

0 0 1 2 3 4 5

1/[S0]

Topic 10.7

KM= 2.5 -1/KM= -0.4 MCMP 304 9/26/11 20

Comparison of Different Vmax At the Same KM

3

2.5

KM= 0.5 -1/Km= -2.0 for all three

VMax= 1.2 1/VMax= 0.83

VMax= 2.1 1/VMax= 0.48

1/v 0

1.5

0.5

VMax= 4.2 1/VMax= 0.24

0 1 2 3 4 5

0 -3 -2 -1

1/[S0]

Topic 10.7

MCMP 304 9/26/11 21

Product Inhibition Revisited

If product P is present in the reaction, it slows the release of P from [EP], thus it inhibits the forward reaction. For unimolecular reactions, this violates the assumptions of the Michaelis-Menton model, so a different model (equation) must be used. Typical kinetic studies avoid this by measuring initial rates starting from substrates only with no product present over time periods where negligible product accumulates. When modeling metabolism in cells, the alternative model must be used that takes product inhibition into account. When using enzymes as part of an industrial process, product inhibition can significantly limit production.
Topic 10.7

MCMP 304 9/26/11 22

Kinetics of Two-substrate Enzyme-catalyzed Reactions Topic 10.8

Topic 10.8

MCMP 304 9/26/11 23

Two-Substrate Reaction Mechanisms

A+B Or: A + B C+D A + B

There are two possible types of mechanisms: Sequential bi-molecular (bi-bi) mechanism (random or ordered) Ping pong bi-bi mechanism Similarities between these mechanisms: Both involve reactions that are described above Differences between these mechanisms: Number of reaction steps and transition states Relatively stable binding of neither, one, or both substrates before formation of a transition state Formation (or not) of a stable reaction intermediate associated with the enzyme or its prosthetic group MCMP 304 9/26/11 24 Topic 10.8

Sequential Mechanism
Sequential bi-molecular mechanism (random or ordered):
B B A A E + A EA ETS* EA E + A E + B EB ETS* EB E + B

Random sequential: Both of the above occur Ordered sequential: Only one of the above occurs, typically because one substrate can bind well, but not the other. Examples include oxidoreductases using NAD+/NADP+ and many kinases. In this mechanism: There is only a single transition state There is no enzyme-bound stable reaction intermediate The enzyme must be able to relatively stably bind one substrate (ordered) or either substrate (random) while waiting to bind the other substrate MCMP 304 9/26/11 25 Topic 10.8