BIOCHEMICAL

ENGINEERING
INTRODUCTION
TO
ENZYME KINETICS
 STRUCTURE
 GLOBULAR PROTEIN ( 3-D
STRUCTURE)
 PRIMARY STRUCTURE
 SECONDARY STRUCTURE
 TERTIARY STRUCTURE
 QUATERNARY STRUCTURE
 ISOENZYMES, ISOZYMES, ISOFORMS
 TYPES AND
NOMENCLATURE
 TRIVIAL NAME: ‘-ase’
 ENZYME COMMISSION NUMBER(EC)
 UNIQUENESS
 High specificity for substrate.
 Operate under ‘mild’ conditions
 High reaction rates.
 Can be regulated.
 Progress of a chemical
reaction
transition state
Free Energy (G)

Activation energy

initial state

∆G ∆G is negative;
reaction goes
final state

Progress of reaction
 Progress of Enzyme reaction
S to P
transition state
Enzyme lowers
activation energy
Free Energy (G)

Activation energy

S
initial state
ES ∆G is negative;
∆G
P reaction goes
final state

Progress of reaction
 How do catalysts work?
Activation
Energy

Catalysts work by stabilizing the transition

state of a chemical reaction, which lowers
the activation energy of the reaction
What sort of rate acceleration
can enzyme provide?
Consider the reaction:

Eact Relative Rate

No Catalyst 18 kcal/mol 1
Pt black 12 kcal/mol 1X10^4
Enzymes 2 kcal/mol 3X10^11

Enzymes are capable of providing astonishing

rate enhancements
 Lets take a look at a real
example!
ATP

Mg(2+)
ATP hydrolysis as an
example
Does an enzyme only catalyze
the forward reaction? NO!

Because the free

energy difference
between reactants and
products of a reaction
and the starting
concentration of each
determines the
direction.
How do enzymes do the
amazing things they do?
 UNIQUE FEATURES OF THE
ENZYMES
 Enzymes increase rate of
reaction but DO NOT change
equilibrium point.
 Enzymes DO NOT supply energy
to reaction, but instead lower
the activation energy
requirement
 Substrate(s) bind at ACTIVE
SITE(S) with BINDING SITE and
CATALYTIC GROUPS
 NOVEL USES OF
ENZYMES
 as monitors of toxic chemical levels
in food and water
 exploitation of enzymes as
electrocatalysts (specific biosensors)
 Enzyme utilisation in formation of
food flavours and aroma compounds
 Enzyme technology in the prevention
of dental cavities
 APPLICATIONS OF
ENZYMES
 Proteas: Clotting and manufacture of
cheese
 Glucose Isomerase: Manufacture of
high-fructose syrups as 'high
sweeteners'
 Glucose oxidase : Analysis of blood
glucose levels
 Pectinases: Juice/Wine clarification
 Amylases: Brewing
 ENZYME SUBSTRATE BINDING
AND SPECIFICITY
 COMPLEMENTARITY
OF STRUCTURES

 STERIC, CHARGE
NEUTRALITY AND
HYDROGEN BOND
FACTORS

 ATP TO MYOSIN, Kd
= 10-13 M
MECHANISM OF CATALYSIS

 Induced fit model.

 Catalysis by bond strain

 Catalysis by proximity and orientation

 Catalysis Involving Proton donors or

acceptors

 Covalent Catalysis
 ENZYME ASSAYS

 Spectrophotometri
c or
radiometric

 Initial rate over 1

min

 Rapid assays
 FAST REACTIONS

 Pre-steady state phase less than 1s

 Rapid mixing

 Estimation of unitary rate constants

STOPPED FLOW
CONTINUOUS FLOW
ULTRA FAST MIXING
CONTINUOUS FLOW
QUENCH FLOW
 RELAXATION METHOD

 Reaction at equilibrium perturbed

 Spectroscopic monitoring

 Temperature, Pressure and

electric field strength

 Reactions with half lives 10-10 s

 Michaelis-Menten
Equation
 Briggs-Haldane equation

 Vmax = K2 [Eo]

 M-M Equation : K2 << K -1

Solution of MM
Equation
1st Approximation

 [E] is small

 [A] = [A]o - [C]


 2 nd
Approximation
 [E] = [E]0 - [B]
 [A] = [A]0


 Multi Substrate
Reactions

 Modified M-M eq
 Types:
 Sequential Reactions
 Ping-Pong Reactions
 Measurement of Vi
 Set up a series of tubes containing graded
concentrations of substrate, [S].
 At time t=0, add a fixed amount of the
enzyme preparation.
 Over the next few minutes, measure the
concentration of product formed.
 If the product absorbs light, we can easily
do this in a spectrophotometer.
 Early in the run, when the amount of
substrate is in substantial excess to the
amount of enzyme, the rate we observe is
the initial velocity Vi.
 Michaelis-Menten plot
 Equation: V =(Vmax [S])
/ (Km + [S])
 Rectangular hyperbola
 Hard to achieve Vmax
 Hard to work out
inhibition patterns

 Convert hyperbolic
curve to a straight line
by doing a reciprocal
plot – Lineweaver-Burk
 Lineweaver-Burk OR
Double Reciprocal plot
 Plot of 1/v against
1/[S]
 Y-intercept: vmax
 X-intercept: Km 1 1 Km 1
 Better estimate of = +
vmax v v max v max [ S ]
 Also useful in
determining the
nature of inhibition
 Other plots
 Eadie-Hofstee plot:
• Plot of v against v/[S]. Km
• Y-intercept: vmax v = v max − v
• X-intercept: Km [S ]

 Hanes-Wolf plot: [ S ] [ S ] Km
• Plot of [S]/v against [S]. = +
• X-intercept: Km v v max v max
3. Y-intercept: vmax
 Case Study:
Characterization of ALP
enzyme activity
Spectrophotometer: Determine Activity of
(PNP absorbs at 410 nm) Alkaline Phosphatase:
 absorbance value
recorded every  0.75 ml 0.4 mM PNPP
second (Lab view) 0.2
 absorbance (A)   M Tris-HCl buffer added
concentration (mM) (3 ml total volume)
 3 ml total volume:
PNPP + buffer (blank)  0.2, 0.3, 0.4, 0.5 ml ALP
, ALP added
 plot absorbance vs.  initial slope A vs. time
time plot
activity (mU/ml)
Michaelis-Menten Modeling:
 0.075, 0.40, 0.75, 1.15, 1.50 ml 0.4 mM
PNPP
 buffer added (3 ml total volume)
 0.2 ml ALP
 initial slope A vs. time  reaction
velocity (mM/min)
0.400 mL Enzyme + 0.75 mL 0.4 mM Substrate

1.4
Absorbance

1.2
y = 0.00472x + 0.04006
1
R2 = 0.99986
0.8
0.6
0.4
0.2
0
0 100 200 300 400
Time (s)
 Lineweaver-Burk plot
Lineweaver-Burke Plot y = 0.3374x + 100.24
 1/v (min/mM) vs. 1/[S] R2 = 0.9586
(mM-1) 160
140
 x-intercept = 120
-308.951 mM-1 = -1/Km 100
80
1/V (min/mM)
60
 Km = 0.00337 mM
40
20
 y-intercept = 100.24 0
min/mM = 1/Vmax -400 -300 -200 -100 -20 0 100 200
-40
 Vmax = 0.00999 1/[s] (1/mM)
mM/min
-1/Km 1/Vmax
 Eadie-Hofstee
 v/[S] (min-1) vs. v
(mM/min) Eadie-Hofstee Plot y = -274.51x + 2.7575
2
R = 0.938
3
 x-intercept = Vmax
2.5
= 0.010045 mM/min

v/[S] (1/min)
2
1.5
 Vmax = 0.010045 1
mM/min
0.5
0
 y-intercept = Vmax/Km 0 0.005 0.01 0.015
= 2.7575 min-1 v (mM/min)

 Km = 0.00365 mM Vmax/Km Vmax

 Hanes-Wolf Plot
 [S]/v (min) vs. [S] Hanes-Woolf Plot y = 97.434x + 0.536
R2 = 0.9995
(mM) 25

20
 x-intercept =
-0.00550 mM = -Km 15

[S]/v (min)
10

 Km = 0.00550 mM 5

0
-0.05 0 0.05 0.1 0.15 0.2 0.25
 y-intercept = 0.536 -5

= Km/Vmax [S] mM

Km/Vmax
-Km
 Vmax = 0.01026
mM/min
 Conclusions
 Hanes-Wolf Model best linearizes Michaelis-Menten
equation
 greatest linear correlation (R2=.9995 )

 Lineweaver-Burk plot does not equally weight all data

points
 exaggerates error at low substrate levels where
measurements are less accurate (slower reaction velocities)
 Another disadvantage is that most experimental
measurements involve relatively high [S] and are thus
crowded toward the left side of the plot.

 The Eadie-Hofstee plot not as error-prone as

Lineweaver-Burke plot
 equal weighting to all data points

 Application of Km:
Hexokinase and

Glucokinase:
Catalyze the first step in glucose metabolism:
Glucose + ATP = Glucose-6-phosphate + ADP
 Normal [ATP] = 1-2 mM. Hence reaction is
independent of [ATP].
 Hexokinase:
found in cells utilizing glucose as an energy
source. Low Km for glucose = 0.1 umol/L so that
it will be saturated at lower glucose levels and
inhibited allosterically by G-6-P product.
provides G-6-P for energy production
• Intracellular glucose levels = 0.2 umol/L.
• Conversely, blood glucose levels = 5 mmol/L, therefore,
hexokinase would not operate efficiently in that
environment at those higher levels.
 Application of Km:
Hexokinase and
Glucokinase:
 Glucokinase – present in liver hepatocytes and
pancreatic cells and plays a physiologic role in
glucose synthesis and storage which takes
place in the liver.
 Provides G-6-P for glycogen synthesis
 The liver is responsive to changes in blood
glucose concentration
 Catalyzes the same reaction but has an > Km for
glucose = 10 mmol/L and is not inhibited by the
product.
 Km glucokinase > Km for hexokinase.
 Glucokinase forms its product only when glucose levels
become higher such as following a meal.
Application of Km:
Hexokinase and
Glucokinase:
 Multisubstrate Reaction
Kinetics
 Reactions with more than one
substrate, of the
A + Btype
→C+D

 Three mechanisms characterized by the

order of substrate addition to the
enzyme and the order of product
release.
 Reaction equations differ depending on
mechanisms r max[ A][ B]
r=
( Kma + [ A])( Kmb + [ B ])
Multisubstrate Reactions
3. Ping-Pong:
 The enzyme binds the first
 Three mechanisms: S and releases the first P
before addition of the
second S
5. Sequential order:  Part of the first S is
Substrate addition transferred to E to form a
and products release modified form F
both in obligate order.  F now binds the second S
and forms the second P
 The chemical
2. Random order: transformation on F is
Substrate addition transferred to second S
and product release is  E is regenerated
in random order.
 Multisubstrate Reaction
Kinetics
 Sequential kinetics can be distinguished
from ping-pong kinetics by initial rate
studies.
 In practice, measure initial rates as a
function of one substrate while holding the
other constant. Then, vary the
concentration of the second substrate and
repeat.
 Lineweaver-Burk analysis should yield a
family of lines that intersect at the left of
the y-axis of the graph.
Multisubstrate Reaction
Kinetics
 What is Enzyme
Inhibition?
 Interference with the enzyme’s
ability to convert substrate to
product - usually a decrease in
velocity OR a complete cessation of
activity.

 Inhibitors bind to the enzyme or

enzyme-substrate complex and
decrease the enzyme activity.
 Why is Enzyme inhibition
important?
 Benefits
 Medicine/drugs
 Regulation of processes in human body

 The other side

 Poisons ( Mustard gas, nerve gas etc)
 Types of Inhibition
 Irreversible (suicide) inhibition
 Reversible inhibition:
 Competitive

 Non-competitive

 Uncompetitive

 Mixed
 Types of Inhibition
Competitive Inhibition
Non Competitive
Inhibition
 Relevant Examples of
Enzyme Inhibition
1)Application in Medicine
 Drug strategy to attack deadly HIV
virus.

 HIV virus-attacks Helper T cells and

destroys immune system.

 Reverse transcriptase enzyme

catalyses production of DNA from
Mechanism of destruction
of immune
cells by HIV virus
 Enzyme reverse transcriptase is a
major target
target site for HIV-fighting drugs.

 Zidovudine (AZT), inhibits reverse

transcriptase and hence in used in
HIV drugs .
More examples of enzyme
inhibition
 Ethanol metabolism in body
EthanolAcetaldehydeAcetic
acid
 Disulfiram(Antabuse) drug inhibits
aldehyde oxidase.
 Accumulation of acetaldehde.
 Respiration Regulation
 Respiration is regulated by feedback
inhibtion

 GlucoseCarbon dioxide
+water+ATP.

 Citrase synthase is inhibited by ATP.

 Required amount of energy is

generated.
 Allosteric Enzymes
 Class of enzymes that bind small,
physiologically important
molecules and modulate
activity.
 The binding molecules—effectors.
 Effectors bind to enzyme at
distinct site,
change is transmitted to
catalytic site.
 Positive and negative effectors.
 EFFECT OF VAROIUS
PARAMETERS
ON ENZYME ACTIVITY

 pH
 Temperature
 Solvent
 pH

 Affects the activity, structural

stability and solubility of the enzyme.

 Only ionic species of a specific

charge causes activity of enzyme.
 -- A

-- B

pH optimum = (pKa1 + pKa2) / 2

pH for optimum activity
•Lipase (pancreas) - 8.0
•Lipase (stomach) - 4.0-5.0
•Invertase - 4.5
 TEMPERATURE EFFECT

 Energetic collisions.

 Number of collisions per unit time.

 Unfolding.
 On reducing temperature, activity can be
restored only to SOME extent.
 DEPENDENCE ON
SOLVENT

 Enzymes work in aqueous solution.

 In organic phase, UNFOLDING occurs

causing deactivation of enzyme.