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
Free Energy (G)

transition state

Activation energy

initial state

G is negative;
reaction goes

final state

Progress of reaction

Progress of Enzyme reaction

S to P
Enzyme lowers
activation energy

Free Energy (G)

transition state

Activation energy

initial state

ES

G is negative;
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:

No Catalyst
Pt black
Enzymes

Eact
Relative Rate
18 kcal/mol
1
12 kcal/mol
1X10^4
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

COMPLEMENTARIT
YOF 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]

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
Better estimate of
vmax
Also useful in
determining the
nature of inhibition

1
1
Km 1

v v max v max [ S ]

Other plots

1.
2.
3.

Eadie-Hofstee plot:
Plot of v against v/[S].
Y-intercept: vmax
X-intercept: Km

Hanes-Wolf plot:
1.
Plot of [S]/v against [S].
2.
X-intercept: Km
3. Y-intercept: vmax

Km
v v max
v
[S ]
[ S ] [ S ] Km

v v max v max

Case Study:
Characterization of ALP
enzyme activity
Determine Activity of

Spectrophotometer:
(PNP absorbs at 410 nm)

absorbance value
recorded every second
(Lab view)
absorbance (A)
concentration (mM)
3 ml total volume:
PNPP + buffer (blank) ,
ALP added
plot absorbance vs.
time

Determine Activity of
Alkaline Phosphatase:

0.75 ml 0.4 mM PNPP 0.2

M Tris-HCl buffer added

(3 ml total volume)

0.2, 0.3, 0.4, 0.5 ml ALP

initial slope A vs. 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)

Lineweaver-Burk plot

1/v (min/mM) vs. 1/[S]

(mM-1)

x-intercept =
-308.951 mM-1 =
-1/Km

Km = 0.00337 mM

y-intercept = 100.24
min/mM = 1/Vmax

Vmax = 0.00999
mM/min

-1/Km

1/Vmax

Eadie-Hofstee

v/[S] (min-1) vs. v (mM/min)

x-intercept = Vmax
= 0.010045 mM/min

Vmax = 0.010045
mM/min

y-intercept = Vmax/Km
= 2.7575 min-1

Km = 0.00365 mM
Vmax/Km

Vmax

Hanes-Wolf Plot

[S]/v (min) vs. [S] (mM)

x-intercept =
-0.00550 mM = -Km

Km = 0.00550 mM

y-intercept = 0.536
= Km/Vmax

Vmax = 0.01026
mM/min

-Km

Km/Vmax

Conclusions

Hanes-Wolf Model best linearizes Michaelis-Menten equation

Lineweaver-Burk plot does not equally weight all data points

greatest linear correlation (R 2=.9995 )

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

angular distortion of error since reaction velocity present on both axes

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 B type
CD

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]

( Kma [ A])( Kmb [ B ])

Multisubstrate Reactions

Three mechanisms:

1.

Sequential order:
Substrate addition and
products release both in
obligate order.

2. Random order:
Substrate addition and
product release is in
random order.

3. Ping-Pong:
The enzyme binds the first
S and releases the first P
before addition of the
second S
Part of the first S is
transferred to E to form a
modified form F
F now binds the second S
and forms the second P
The chemical
transformation on F is
transferred to second S
E is regenerated

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 enzymes 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 viral-RNA.

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

EthanolAcetaldehydeAcetic
acid
Disulfiram(Antabuse) drug inhibits
aldehyde oxidase.

Accumulation of acetaldehde.

Respiration Regulation

Respiration is regulated by feedback

inhibtion

GlucoseCarbon 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 moleculeseffectors.
Effectors bind to enzyme at distinct site,
change is transmitted to catalytic site.
Positive and negative effectors.
K type Enzyme and V type enzyme.

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.