ENZYME KINETICS

By: Engr. Vera Marie L. Lanaria

ChE Department CIT University

Kinetics of Enzyme Reactions

deals with the rate of enzyme reaction and how it is affected by various chemical and physical conditions it provides information about the basic mechanism of the enzyme reaction and other parameters that characterize the properties of the enzyme rate equations can be applied in calculating reaction time, yields, & optimum economic conditions needed in designing bioreactors

Let S be the substrate (reactant) E be the enzyme P be the product A simple reaction would be: S+EP
Rate of reaction can be expressed in terms of: r = vs = - dS/dt or: vp = dP/dt

Victor Henri (1902, a French physical chemist) proposed a quantitative theory of enzyme kinetics and formulated the rate equation: v = vmax S KM + S In 1913, Leonor Michaelis (German biochemist) and Maud Menten (Canadian physician) continued the work of Henri in which later on it becomes the MichaelisMenten model

Michaelis-Menten Model

Lock-and-Key Model (Emil Fischer 1894)

Induced-fit Model
(Daniel Koshland 1958)

Derivation of Reaction Rate Equation

Assumptions: The total enzyme concentration stays constant during reaction, that is, CEo = CES + CE The amount of an enzyme is very small compared to the amount of substrate; so the formation of enzyme-substrate complex does not significantly deplete the substrate.

The product concentration is so low that product inhibition may be considered negligible.

Linear Forms of MichaelisMenten Equation

Langmuir plot (or Hanes Woolf plot) Lineweaver-Burk plot Eadie-Hofstee plot

Langmuir Plot

Lineweaver-Burk Plot

Eadie-Hofstee Plot

Sample Problem:
From a series of batch runs with a constant enzyme concentrations, the following initial rate data were obtained as a function of initial substrate concentration. (Refer to the next slide for the data.) Evaluate the Michaelis-Menten kinetic parameters by employing the 3 linear forms or plots. In evaluating the parameters do not include data points which deviate systematically from the Michaelis-Menten model.

S (mmol/L) 1 2 3 5 7 10 15 20

v (mmo/L-min) 0.20 0.22 0.30 0.45 0.41 0.50 0.40 0.33

Solution:
Examination of the data reveals that as the substrate concentration (S) increased up to 10 mmo/L, the rate increased. However, the further increases in the S to 15 mmol/L, the initial reaction rate decreased. This behavior may be due to substrate or product inhibition. Since the Michaelis-Menten equation does not incorporate the inhibition effects, thus these two data points will be included.

0.60 0.50 0.40 0.30 0.20 0.10 0.00 0 5 10 S (mmo/L) 15 20 25

v (mmol/Lmin)

Langmuir Plot
25
S/v (min)

y = 1.5866x + 4.6417 R2 = 0.9497

20 15 10 5 0 0 2 4 6 S (mmol/L 8 10 12

From the line equation: y = 1.5866x + 4.6417

slope = 1/vmax = 1.5866 vmax = 1/1.5866 vmax = 0.63 min-1 y-intercept = KM/vmax = 4.6417 KM = (4.6417)(0.63) KM = 2.92 mmol/Lmin2

Lineweaver-Burk Plot
6
1/v

y = 3.4575x + 1.945 R2 = 0.8463

4 2 0 0 0.2 0.4 0.6 1/S 0.8 1 1.2

From the line equation: y = 3.4575x + 1.945

y-intercept = 1/vmax = 1.945 vmax = 1/1.945 vmax = 0.514 min-1 slope = KM/vmax = 3.4575 KM = 3.4575(0.514) KM = 1.78 mmol/Lmin2

Eadie-Hofstee Plot
0.60 0.50 0.40 0.30 0.20 0.10 0.00 0 0.05 0.1 v/S 0.15

y = -1.8923x + 0.5386 2 R = 0.6618

0.2

0.25

From the line equation: y = -1.8923x + 0.5386

y-intercept = vmax = 0.5386 vmax 0.54 min-1 slope = -KM = -1.8923 KM = 1.8923 KM 1.89 mmol/Lmin2

Enzyme Reactor with Simple Kinetic

Bioreactor is a device/equipment within which biochemical transformation are caused by the action of enzyme or living cells Classifications of bioreactor: 1) Batch 2) Steady-State Plug-Flow Reactor (PFR) 3) Continuous Stirred-Tank Reactor (CSTR)

Batch Reactor
is normally equipped with agitator pH is maintained by using either a buffer solution or a pH controller an ideal batch reactor is assumed to be well mixed so that the contents are uniform in composition at all times

Reaction Mechanism:
- dS = vmax S dt KM + S rearranging & integrating: -(KM+S).dS/S = vmax.dt passing the limits: at t=0 ; S = So at t=t ; S = S - KM ln(S/So) (S So) = vmaxt KM ln(So/S) + (So S) = vmaxt

PFR Reactor (or Tubularflow Enzyme Reactor)

the substrate enters one end of a cylindrical tube which is packed with immobilized enzyme and the product stream leaves at the other end properties of flowing stream will vary in both longitudinal and radial directions since there is no agitator used

 since the variation in the radial direction is small compared to that in the longitudinal direction, its called plug-flow reactor if PFR is operated at steady-state, the properties will be constant with respect to time equation in batch reactor can be applied to an ideal steady-state PFR, however, the time, t, should be replaced with the residence time, So S = -KM + vmax . ln(So/S) ln(So/S)

CSTR
is an ideal reactor which is based on the assumption that the reactor contents are well mixed continuous operation can increase the productivity significantly by eliminating the downtime easy to automate

 substrate balance can be set up as follows: Input - Output + Generation = Acc. F(So) - F(S) + rsV = V(dS/dt) where: F = flow rate V = volume of the reactor rs = rate of substrate consumption but for steady-state CSTR, the concentration of substrate should be constant, thus dS/dt = 0

and if Michaelis-Menten equation can be used for the rate of substrate consumption, then the equation can be arranged as: F = D = 1/ = vmax S . V (So S)(KM + S) where: D = is known as dilution rate (Note: Its common in biochemical reaction to use the term dilution rate, than the term residence time.) S = -KM + (vmaxS)(So S)

Inhibition of Enzyme Reaction

Inhibitor can decrease the rate of reaction either competitively, non-competitively, partially competitively, or mixed

Other Factors that influences Enzyme Activity

temperature pH effect of shear