Chapter 2

Enzyme Kinetics

Prepared By Jemal A (MSc): 2024

 Learning objectives

 The main objective is to explain how enzymes work to speed up chemical

reactions and the study of the rates of enzyme – catalyzed chemical
reactions.
 Self-test:

1) What is enzymes? Explain major functions of enzymes in a living systems.

2) Explain the difference between biological and non-biological catalyst.

3) Define enzyme kinetics.

 Outline:

 Introduction of Enzyme

 Commercial Applications of Enzyme

 Simple Enzyme Kinetics

 Evaluation of Kinetic Parameters

 Enzyme Reactor with simple kinetics

 Inhibition of Enzyme Reactions

 Other Influences on Enzyme Activity

 Enzymes are biological catalyst that are protein molecules in nature.
 They are produced by living cells (animals, plant and microorganisms) and absolutely
essential as catalysts in biochemical reactions.
 Almost every reaction in a cell requires the presence of a specific enzyme.
 A major function of enzymes in a living systems is to catalyze the making and
breaking of chemical bonds.
 Therefore, like any other catalysts, they increase rate of reaction without themselves
undergoing permanent chemical changes.
 Enzyme catalytic ability is due to its particular protein structure.
 A specific chemical reaction is catalyzed at a small portion of the surface of an enzyme
called active site.

 Some physical and chemical interactions occur at this site.

 Enzymes as biological catalysts

 Why enzyme reactions are different from chemical reactions?

 Difference between biological and non-biological catalyst
Biological Non biological

Specific and selective: there is specific type of catalyst for a Non Specific and selective: many reactions can be catalyzed
specific type of reaction. Adv.:-minimize down stream by a single catalyst or a reaction may be catalyzed by wide
processing cost and result high desired product amount. range of catalysts.

Efficient in small amount ( high turn over) Non-efficient in small amount

Enzymatic rate of reaction is usually much faster than Reaction directed by non-biological catalysts is slower than
reaction directed by non-biological catalysts. enzymatic reaction

Lower activation energy (Ea) Higher activation energy (Ea)

Highly sensitive to processing conditions (advantages:- low Less sensitive: can work at high T oc & P. So, require high
energy requirement) energy & special resistant material.
 Nomenclature of Enzymes
 Originally enzymes were given non descriptive names such as:

• rennin: curding of milk to start cheese making process.

• pepsin: hydrolyzes proteins at acidic pH.

• trypsin: hydrolyzes proteins at mild alkaline pH.

 The nomenclature was later improved by adding suffix -ase to the name of the substrate
with which the enzymes functions, or the reaction that is catalyzed.
 For Example:
 Name of substrate + -ase
• 𝜶 – amylase starch → glucose + maltose + oligosaccharides
• lactase lactose → glucose + galactose
• lipase fat → fatty acids + glycerol
• maltase maltose → glucose
• urease urea + H2O → 2NH3 + CO2
• cellobiase cellobiose → glucose
 Reaction which is catalyzed + -ase
• alcohol dehydrogenase ethanol + NAD+ ↔ acetaldehyde + NADH2

• glucose isomerase glucose ↔ fructose

• glucose oxidase D-glucose + O2 + H2O → gluconic acid

• lactic acid dehydrogenase lactic acid → pyruvic acid

 As more enzymes were discovered, this system generated confusion and resulted in the formation of a new
systematic scheme by the International Enzyme Commission in 1964.

 The new system categorizes all enzymes into six major classes depending on the general type of chemical
reaction which they catalyze.

 Each main class contains subclasses, sub-sub-classes and sub-sub-sub-classes. Therefore, each enzyme can
be designated by a numerical code system.
 Table 2.1 Potential outline of the systematic classification of enzymes

 Example:
o Reaction: CH2CH2OH + NAD – CH3CHO + NADH + H+
o Systematic Name: alcohol NAD oxidoreductase (1.1.1.1.)
o Trivial Name: alcohol dehydrogenase.
 Commercial Applications of Enzymes
 Enzymes were used in
 Food industry:
 making sweets from starch by converting starch to monomer unit (HFCS in soft drinks).
 clotting milk to produce cheese .
 brewing soya sauce
 dairy product
 Baking and drinking industry
 Sugar refining: hydrolytic enzymes such as Dextranase and Amylase.
 Alkaline protease is
 added to laundry detergent as cleaning aid.
 also used for meat tenderizer & cheese making.
 The enzymes produced commercially can be classified into three major categories.

1) Industrial enzymes, such as amylases, proteases, glucose isomerase, lipase, catalases, and
penicillin acylases

2) Analytical enzymes, such as glucose oxidase, galactose oxidase, alcohol dehydrogenase,

hexokinase, muramidase, and cholesterol oxidase

3) Medical enzymes, such as asparaginase, proteases, lipases, and streptokinase

 α-amylase, glucoamylase, and glucose isomerase serve mainly to convert starch into high-fructose corn
syrup (HFCS), as follows:
𝜶−𝒂𝒎𝒚𝒍𝒂𝒔𝒆 𝑮𝒍𝒖𝒄𝒐𝒂𝒎𝒚𝒍𝒂𝒔𝒆 𝑮𝒍𝒖𝒄𝒐𝒔𝒆 𝒊𝒔𝒐𝒎𝒆𝒓𝒂𝒔𝒆
𝐶𝑜𝑟𝑛 𝑠𝑡𝑎𝑟𝑐ℎ 𝑇ℎ𝑖𝑛𝑛𝑒𝑑 𝑠𝑡𝑎𝑟𝑐ℎ 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 𝐻𝐹𝐶𝑆
 Simple enzyme kinetics
 Enzyme kinetics deals with the rate of enzyme reaction and how it is affected by various chemical
and physical conditions.

 Kinetic studies of enzymatic reactions provide information about the basic mechanism of the enzyme
reaction and other parameters that characterize the properties of the enzyme.

 The rate equations developed from the kinetic studies can be applied in calculating reaction time,
yields, and optimum economic condition, which are important in the design of an effective
bioreactor.
 Assume that a substrate (S) is converted to a product (P) with the help of an enzyme (E) in a
reactor as ;
𝑬
𝑺 𝑷

 Concentration of S is decreased with respect to time.

 Concentration of P is increased with respect time and reach a max. value as follows:

Figure: The change of product and substrate concentrations with respect to time.
 Rate of reaction can be expressed in terms of either change of the substrate CS or product
concentrations CP as follows:
−𝒅𝑪𝒔 𝒅𝐂𝒑
rs = or rp =
𝒅𝒕 𝒅𝒕

 In order to understand the effectiveness and characteristics of an enzyme reaction, it is

important to know how the reaction rate is influenced by reaction conditions such as substrate,
product, and enzyme concentrations.
 If we measure the initial reaction rate at different levels of substrate and enzyme concentrations, we
obtain a series of curves.

 Figure: The effect of substrate concentration on the initial reaction rate.

 From these curves we can conclude the following:

1) The reaction rate is proportional to the substrate concentration (that is, first-order reaction)
when the substrate concentration is in the low range.

2) The reaction rate does not depend on the substrate concentration when the substrate
concentration is high, since the reaction rate changes gradually from first order to zero order as
the substrate concentration is increased.

3) The maximum reaction rate, 𝒓𝒎𝒂𝒙 is proportional to the enzyme concentration within the range
of the enzyme tested.
 Henri observed this behavior in 1902 and proposed the rate equation,

𝒓𝒎𝒂𝒙 𝑪𝑺
𝒓𝒑 =
𝑲𝑴 + 𝑪𝑺

where 𝒓𝒎𝒂𝒙 and 𝑲𝑴 are kinetic parameters which need to be experimentally determined.

 It expresses the three preceding observations fairly well.

 The rate is proportional to Cs (first order) for low values of Cs , but with higher values of Cs, the rate
becomes constant (zero order) and equal to 𝑟𝑚𝑎𝑥 .
 (in a fixed period of time)
+

P
E
↓
S

8
8
7

Substrate (mmole)
6
6
5

4
4
3
2

2
1
0

0
0
80

60

40

20
Product
Increase Substrate Concentration

 An Example for Enzyme Kinetics (Invertase)
1) Use predefined amount of Enzyme → E
2) Add substrate in various concentrations → S (x axis)
3) Measure Product in fixed Time (P/t) → ro (y axis)
4) (x, y) plot get hyperbolic curve, estimate → r max
5)When y = 1/2 rmax, calculate x ([S]) → Km
Double reciprocal r max
1
ro ro
Direct plot
1/2
-1
Km 1
rmax 1/S S
Km
 Steady State Theory

E + S E S E +P
 In steady state, the production and consumption of the transition state proceed at the same rate.
 So the concentration of transition state keeps a constant.
 Constant ES Concentration at Steady State

S
P
Concentration

ES
E

Reaction Time
 Brown (1902) proposed that an enzyme forms a complex with its substrate.

 The complex then breaks down to the products and regenerates the free enzyme.

 The mechanism of one substrate - enzyme reaction can be expressed as

eq. (1)

eq. (2)
 One of the original theories to account for the formation of the enzyme-substrate complex is the
"lock and key" theory.

 The main concept of this hypothesis is that there is a topographical, structural compatibility
between an enzyme and a substrate.

 Figure: Lock and key theory for the enzyme – substrate complex.
 The reaction rate equation can be derived from the preceding mechanism based on the following
assumptions:

1 .The total enzyme concentration stays constant during the reaction.

𝐶𝐸𝑂 = 𝐶𝐸𝑆 + 𝐶𝐸

2. The amount of an enzyme is very small compared to the amount of substrate. Therefore, the
formation of the enzyme - substrate complex does not significantly deplete the substrate.

3. Product concentration is so low that product inhibition is considered to be negligible.

 In addition to the preceding assumptions, there are three different approaches to derive the rate equation:

1) Michaelis - Menten approach, 1913:

 It is assumed that the product releasing step, eq. (2), is much slower than the reversible reaction, eq. (1), and the slow step
determines the rate, while the other is at equilibrium. This is an assumption which is often employed in heterogeneous
catalytic reactions in chemical kinetics.

2) Briggs - Haldane approach, 1925:

 The change of the intermediate concentration with respect to time is assumed to be negligible, i.e., d(CES)/dt = 0. This is
also known as the pseudo-steady-state (or quasi-steady-state) assumption in chemical kinetics and is often used in
developing rate expressions in homogeneous catalytic reactions.

3) Numerical solution: Solution of the simultaneous differential equations developed from Eq. (1) and (2) without
simplification.
 In the case Michaelis-Menten Approach, if the slower reaction, Eq. (2), determines the overall
rate of reaction , the rate of product formation and substrate consumption is proportional to the
concentration of the enzyme-substrate complex as:

eq. (3)

 Unless otherwise specified, the concentration is expressed as molar unit, such as kmol /m3 or mol/L.

 The concentration of the enzyme-substrate complex CES in Eq. (3), can be related to the substrate concentration Cs and
the free-enzyme concentration CE from the assumption that the first reversible reaction Eq. (1) is in equilibrium.

eq. (4)
 By substituting Eq. (4) into Eq. (3), the rate of reaction can be expressed as a function of CS and CE,
of which CE cannot be easily determined.

 If we assume that the total enzyme contents are conserved, the free-enzyme concentration CE can
be related to the initial enzyme concentration CE0

eq. (5)
 Put eq. (4) into eq. (5) for CE and rearranging for CES.
Then,
eq. (6)
 Put eq. (6) into eq. (3) which result for final rate equation as

eq. (7),

which is known as Michaelis – Menten equations.

 KM is known as the Michaelis constant.

 In the Michaelis-Menten approach, KM is equal to the dissociation constant, K1 or the reciprocal

of equilibrium constant, Keq as
 The unit of KM is the same as CS (kmol/m3).

 When KM is equal to Cs, r is equal to one half of rmax according to Eq. (7).

 Therefore, the value of KM is equal to the substrate concentration when the reaction rate is half of
the maximum rate, 𝑟𝑚𝑎𝑥 .

 KM is an important kinetic parameter because it characterizes the interaction of an enzyme with a

given substrate. Thus, a small value of Km means a strong interaction b/n the substrate and the
enzyme.
 Another kinetic parameter in eq. (7) is the maximum reaction rate, rmax, which is proportional to the
initial enzyme concentration,𝐶𝐸0 . The main reason for combining two constants k3 and CEO, into one lumped
parameter, rmax is due to the difficulty of expressing the enzyme concentration in molar unit (i.e. because of
difficulties in determining enzyme purity).

 Whatever unit is adopted for CEO, the unit for k3CEO, should be the same as r, i.e. kmol/m3s.

o The Michaelis-Menten equation is analogous to the Langmuir isotherm equation,

𝐶𝐴
𝜃=
𝐾 + 𝐶𝐴

where 𝜃 is the fraction of the solid surface covered by gas molecules and K is the reciprocal of the adsorption
equilibrium constant.
 In case of Briggs-Haldane approach, again from eq. (1) and (2) above, the rate of product
formation or substrate consumption are

eq. (8)

eq. (9)

 Assume that the change of CES with time, dCES /dt, is negligible compared to that of CP or CS.
eq. 10
 Again, if we assume that the total enzyme contents are conserved,

eq. (11)
 Put eq. (10) into eq. (11) for CE and rearrange for CES

eq. (12)

 Put eq. (12) into eq. (3 or 8) results

eq. (13)
 Question#1

 Derive rate equation for the following glucose conversion to fructose by glucose isomerase reaction;
use

A. Micheales-Menten Approach;

B. Briggs-Haldane Approach.

N.B. The slow product releasing step is also reversible.

 Question#2
k1
E+S ES
k2

k3
(ES)1 (ES)2
k4

k5
(ES)2 E+P

 Derive rate law for this two intermediate enzyme substrate reaction.
 Numerical Solution

 From the mechanism described by Eqs. (1) and (2), three rate equations can be written for 𝑪𝑷 , 𝑪𝑬𝑺
and 𝑪𝑺 as

With
𝑪𝑬𝟎 = 𝑪𝑬 + 𝑪𝑬𝑺

 These equations can be solved simultaneously without simplification.

 Among many software packages that solve simultaneous differential equations, Advanced Continuous Simulation Language
(ACSL, 1975) is very powerful and easy to use.

 The heart of ACSL is the integration operator, INTEG, i.e. 𝑅 = 𝐼𝑁𝑇𝐸𝐺 𝑋, 𝑅0

implies

𝑅 = 𝑅0 + 𝑋𝑑𝑡
0

 Example:
𝑡
𝐶𝑃 = 𝐶𝑃0 + 𝑘 𝐶 𝑑𝑡
0 3 𝐸𝑆

which can be written in ACSL as

CP = INTEG(K3*CES,CP0)
 Evaluation of Kinetic Parameters
 In order to estimate kinetic parameter, series of batch run with different levels of substrate concentration is needed.

 The initial reaction rate can be calculated as function of initial substrate concentration.

 The most straightforward way is to plot r vs Cs.

 The asymptote for r will be 𝒓𝒎𝒂𝒙

 KM is equal to Cs when r = 𝟎. 𝟓 ∗ 𝒓𝒎𝒂𝒙

 However, this is an unsatisfactory plot in estimating 𝒓𝒎𝒂𝒙 and 𝑲𝑴 because it is difficult to estimate asymptotes accurately
and also difficult to test the validity of the kinetic model.
 Therefore, the Michaelis - Menten equation is usually rearranged so that the results can be
plotted as straight line.

𝑪𝑺 𝑲𝑴 𝑪𝑺
= + (Eq.14) → Langmuir Plot
𝒓𝒑 𝒓𝒎𝒂𝒙 𝒓𝒎𝒂𝒙

𝟏 𝟏 𝑲𝑴 𝟏
= + (Eq.15) → Lineweaver-Burk Plot
𝒓𝒑 𝒓𝒎𝒂𝒙 𝒓𝒎𝒂𝒙 𝑪𝑺

𝒓𝒑
𝒓𝒑 = 𝒓𝒎𝒂𝒙 − 𝑲𝑴 (Eq.16) → Eadie-Hofstee Plot
𝑪𝑺
 Equation 14, is called as Langmuir plot which result in straight line with 1/rmax slope and KM/rmax intercept.

KM = 10, rmax = 5

 The 15 equation is Lineweaver-Burk plot with slope of KM /rmax and intercept 1/rmax

KM = 10, rmax = 5
 The 16 equation is Eadie-Hofstee plot with slope of –KM and intercept r max.

KM = 10, rmax = 5
 In conclusion, the values of Michaelis-Menten kinetic parameters, rmax and KM can be estimated as follows;

 Make a series of batch runs with different levels of substrate concentration at a constant initial enzyme
concentration and measure the change of product or substrate concentration with respect to time.

 Estimate the initial rate of reaction from the Cs or Cp versus time curves for different initial substrate
concentrations.

 Estimate the kinetic parameters by plotting one of the three plot explained in this section or a non-linear
regression technique.

 All of the above treatments are applicable only to unimolecular irreversible enzyme reactions.
  Example:
 From a series of batch runs with a constant enzyme concentration, the following initial rate data
were obtained as a function of initial substrate concentration.

 Evaluate the michaelis-Menten kinetic parameter by employing the three method, Langmuir,
Lineweaver-Burk plot and Eadie-Hofstee plot.
 Solutions:

 Estimated values of Michaelis - Menten kinetic parameters:

 Enzyme Reactor with Simple Kinetics

¬ A bioreactor is a device within which biochemical transformation are caused by the action of enzyme or living cells.

 The bioreactor employing enzymes an enzyme reactor to distinguish it from the bioreactor which employs living cells,

the fermenter.

 Batch Or Steady State Plug-flow Reactor

 The simplest reactor configuration for any enzyme reaction is the batch mode.

 A batch enzyme reactor normally equipped with an agitator to mix the reactant and the pH of the reactant is

maintained by employing 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.
 Figure (a) Schematic diagram of a batch stirred – tank reactor

 Assume that an enzyme reaction is initiated at t = 0 by adding enzyme and the reaction mechanism can be represented by
Michaelis - Menten equation:

Eq.(17)
 An equation expressing the change of the substrate concentration with respect to time can be obtained by integrating
equation (17) as follows:

eq. (18)

and
𝑪𝑺𝒐 Eq. (19)
𝑲𝑴 𝒍𝒏 + 𝑪𝑺𝒐 − 𝑪𝑺 = 𝒓𝒎𝒂𝒙 𝒕
𝑪𝑺

 This equation shows how Cs is changing with respect to time. With known value of r max and KM, the change of Cs with
time in a batch reactor can be predicted from this equation.
 Plug-flow/Or Tubular-flow Enzyme Reactor:

Fig. (b) Schematic diagram for a plug-flow reactor

 In this 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.

 The long tube and lack of stirring device prevents complete mixing of the fluid in the tube.

 Therefore, the properties of the flowing stream will vary in both longitudinal and radial directions.
 If a plug-flow reactor is operated at steady state, the properties will be constant with respect to time.

 The ideal plug-flow enzyme reactor can approximate the long tube, packed-bed, and hollow fiber, or multistage reactor.

 Eq. (19) can also be applied to an ideal steady-state plug-flow reactor, even though the plug-flow reactor is operated in
continuous mode.

 However, time t should be replaced with the residence time 𝜏 in the plug-flow reactor.

 Rearranging equation(19) results in the following useful linear equation:

𝑪𝑺𝒐 − 𝑪𝑺 𝒓𝒎𝒂𝒙 𝝉
= −𝑲𝑴 + Eq.(20)
𝐥𝐧(𝑪𝑺𝒐 /𝑪𝑺 ) 𝐥𝐧(𝑪𝑺𝒐 /𝑪𝑺 )
 Continues Stirred - Tank Reactor

Figure: Schematic diagram of a continuous stirred-tank reactor (CSTR).

 CSTR is an ideal reactor which is based on the assumption that the reactor content are well mixed.

 Therefore, the concentrations of the various components of the outlet stream are assumed to be the same as the
concentrations of these components in the reactor.

 Continuous operation of the enzyme reactor can increase the productivity of the reactor significantly by eliminating the
downtime. It is also easy to automate in order to reduce labor costs.
 The substrate balance of a CSTR can be set up as follows:
𝑰𝒏𝒑𝒖𝒕 − 𝑶𝒖𝒕𝒑𝒖𝒕 + 𝑮𝒆𝒏𝒆𝒓𝒂𝒕𝒊𝒐𝒏 = 𝑨𝒄𝒄𝒖𝒎𝒖𝒍𝒂𝒕𝒊𝒐𝒏

𝒅𝑪𝑺
𝑭𝑪𝑺𝟎 − 𝑭𝑪𝑺 + 𝒓𝑺 𝑽 = 𝑽 Eq. (21)
𝒅𝒕

where

 F is the flow rate and

 V is the volume of reactor contents,

 rs is the rate of substrate consumption for the enzymatic reaction while

𝒅𝑪𝒔
 𝒅𝒕 is the change of the substrate concentration in the reactor.

 N.B: In Eq. (21), 𝒓𝑺 = 𝒅𝑪𝒔 𝒅𝒕 when F = 0, which is the case in batch operation.
 For the steady - state CSTR, the substrate concentration of reactor should be constant.
𝒅𝑪𝒔
𝒅𝒕 = 𝟎
 If the Michaelis - Menten equation can be used for the rate of substrate consumption (𝒓𝑺 ), Eq. (21) can be rearranged as:

𝑭 𝟏 𝒓𝒎𝒂𝒙 𝑪𝑺
=𝑫= = Eq. (22)
𝑽 𝝉 (𝑪𝑺𝒐 −𝑪𝑺 )(𝑲𝑴 +𝑪𝑺 )

where D is known as dilution rate, and is equal to the reciprocal of the residence time (𝜏).
 Eq. (22) can be rearranged to give the linear relationship:

Eq. (23)
 Michaelis-Menten kinetic parameters can also be estimated by running a series of steady-state CSTR runs with

various flow rates and plotting Cs versus (Cs𝝉) / (CSo - Cs).

 Another approach is to use the Langmuir plot (Cs/r vs Cs) after calculating the reaction rate at different flow rates.

 The reaction rate can be calculated from the relationship:

r = F (Cso - Cs)/V
 Inhibition of Enzyme Reactions

 A modulator (or effector) is a substance which can combine with enzymes to alter their catalytic activities.

 An inhibitor is a modulator which decreases enzyme activity.

 It can decrease the rate of reaction either competitively, noncompetitively, or partially competitively.
 Competitive Inhibition

 Since a competitive inhibitor has a strong structural resemblance to the substrate, both the inhibitor and substrate
compete for the active site of an enzyme.

 The formation of an enzyme-inhibitor complex reduces the amount of enzyme available for interaction with the
substrate and, as a result, the rate of reaction decreases.

 A competitive inhibitor normally combines reversibly with enzyme.

 Therefore, the effect of the inhibitor can be minimized by increasing the substrate concentration, unless the substrate
concentration is greater than the concentration at which the substrate itself inhibits the reaction.

 The mechanism of competitive inhibition can be expressed as follows:

eq. (24)

 If the slower reaction, the product formation step, determines the rate of reaction according to the Michaelis - Menten
assumption, the rate can be expressed as:

eq.(25)
 The enzyme balance gives

eq. (26)

 From the two equilibrium reactions,

eq. (27)

where KS and KI are dissociation constants which are the reciprocal of the equilibrium constants.
 Combining the preceding four equations to eliminate CE, CES and CEI yields
𝐶𝐸𝑜 𝐶𝑆
𝐶𝐸𝑆 =
𝐶𝑆 + 𝐾𝑆 (1 + 𝐾𝐶𝐼 )
𝐼

𝒓𝒎𝒂𝒙 𝑪𝑺 𝐶𝐼
𝒓𝑷 = where 𝐾𝑀𝐼 = 𝐾𝑆 1 + Eq. (28)
𝑲𝑴𝑰 + 𝑪𝑺 𝐾𝐼

 Therefore, since KMI > KS, the reaction rate decreases due to the presence of inhibitor.

 r max is not affected by the presence of competitive inhibitor.

 However, a larger amount of substrate is required to reach the maximum rate.

 Noncompetitive inhibitor

 This is interact with enzymes in many different ways.

 They can bind to enzymes reversibly or irreversibly at the active site or at some other region.

 In any case the resultant complex is inactive.

 Mechanism of noncompetitive inhibition can be expressed as follows:

 eq. (29)

 Since substrate and inhibitor do not compete for a same site for the formation of enzyme-substrate or enzyme-inhibitor
complex, we can assume that the dissociation constant for the first equilibrium reaction is the same as that of the third
equilibrium reaction, as

Similarly,
 As shown in the previous section, the rate equation can be derived by employing the Michaelis - Menten approach as
follows:
𝑟𝐼, 𝑚𝑎𝑥 𝐶𝑆 𝑟𝑚𝑎𝑥
𝑟𝑃 = where 𝑟𝐼, 𝑚𝑎𝑥 =
𝐶𝑆 + 𝐾𝑆 1 + 𝐾𝐶𝐼
𝐼

 Therefore, the maximum reaction rate will be decreased by the presence of a noncompetitive inhibitor, while the
Michaelis constant Ks will not be affected by the inhibitor.
 Figure: inhibition of enzyme reaction
 Figure: The effect of inhibitors as seen in the Langmuir and Lineweaver-Burk plots: (a) competitive, (b) noncompetitive.
 Several variations of the mechanism for noncompetitive inhibition are possible.

• One case is when the enzyme-inhibitor-substrate complex can be decomposed to produce a product and the
enzyme inhibitor complex.

• This mechanism can be described by adding the following slow reaction to Eq. (29).

• This case is known as partially competitive inhibition.

 Exercise:
o Derivate the rate equation for partially competitive inhibition.
 Other Influences On Enzyme Activity

 The rate of an enzyme reaction is influenced by various chemical and physical conditions.

 Some of the important factors are the

 concentration of various components (substrate, product, enzyme, cofactor, and so on),

 pH,

 temperature, and

 shear.
 Effect of pH
 The rate of an enzyme reaction is strongly influenced by the pH of the reaction solution both in vivo and in vitro.
 The typical relationship between the reaction velocity and pH shows a bell-shaped curve Figure below.
 The optimum pH is different for each enzyme.
For Example:
 pepsin from the stomach has pH: 2 - 3.3, amylase, from saliva, is 6.8.
 Chymotrypsin, from the pancreas, has an optimum pH in the mildly alkaline region between 7 and 8.
 The reason that the rate of enzyme reaction is influenced by pH can be explained as follows:
1) Enzyme is a protein which consists of ammo acid residues (i.e., amino acids minus water).

2) The amino acid residues possess basic, neutral, or acid side groups which can be positively or negatively charged at any
given pH.
 Let's consider one acidic amino acid, glutamic acid, which is acidic in the lower pH range.
 As the pH is increased, glutamic acid is ionized as which can be expressed as

 In equilibrium,

When CA- = CA, pH is equal to pK. For glutamic acid, pK = 4.5.

 On the other hand, an amino acid, lysine, is basic in the range of higher pH value. As the pH is decreased, lysine is
ionized as

 Similarly, the pK value of lysine is 10.0, at which half of the residues are ionized.

3. An enzyme is catalytically active when the amino acid residues at the active site each possess a particular charge.

 Therefore, the fraction of the catalytically active enzyme depends on the pH.
 Effect of Temperature
• The rate of a chemical reaction depends on the temperature according to Arrhenius equation as

𝒌 = 𝑨𝒐 𝒆−𝑬𝒂 𝑹𝑻

where 𝐴𝑜 = frequency factor (constant); k = rate constant.

• Consequently, when In k is plotted versus 1/T, a straight line with slope − 𝐸𝑎 𝑅𝑇 is obtained.

• An increase in the temperature increases the rate of reaction, since the atoms in the enzyme molecule have greater energies
and a greater tendency to move.

• However, the temperature is limited to the usual biological range. As the temperature rises, denaturation processes
progressively destroy the activity of enzyme molecules.
 Effect of Shear
 Enzymes had been believed to be susceptible to mechanical force, which disturbs the elaborate shape of an enzyme
molecule to such a degree that denaturation occurs.

 The mechanical force that an enzyme solution normally encounters is fluid shear, generated either by flowing fluid,
the shaking of a vessel, or stirring with an agitator.

𝒅𝒖 𝒅𝒖
𝒔𝒉𝒆𝒂𝒓 𝒔𝒕𝒓𝒆𝒔𝒔, 𝝉 = 𝝁 𝒘𝒉𝒆𝒓𝒆 𝒔𝒉𝒆𝒂𝒓 𝒓𝒂𝒕𝒆, 𝜸 =
𝒅𝒚 𝒅𝒚

 The effect of shear on the stability of an enzyme is important for the consideration of enzyme reactor design, because
the contents of the reactor need to be agitated or shook in order to minimize mass transfer resistance.
 Practice Exercise: From James Lee Reference Book

 Question No:- 2.5, 2.8, 2.9, 2.10, 2.19

End Of Chapter Two.

Thank You!

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