ENZYME KINETICS

EFFECT OF PH, TEMPERATURE, ENZYME AND SUBSTRATE

CONCENTRATION ON SALIVARY AMYLASE
INTRODUCTION
DEFINITION OF TERMS
ENZYME SALIVARY AMYLASE SUBSTRATE

- Biomolecule - Most common - Molecule that binds

composed of member of the to an enzyme’s
proteins in a tertiary glycoside hydrolase active site
globular family of enzymes
conformation - Hydrolyzes
- Acts a catalyst for a-1,4-glycosidic
metabolic reactions bonds for
in the body breakdown of
sugars
DEFINITION OF TERMS

ACHROMIC POINT Michaelis-Menten

- Point wherein - Point wherein

amylase-starch amylase-starch
reaction does not reaction does not
cause a color cause a color
change upon the change upon the
addition of iodine addition of iodine
FURTHER DISCUSSION OF ENZYME KINETICS
- Enzymes provide a separate pathway with lower activation energy for
biochemical reactions to occur.
- Different factors affect the rate of enzyme activity like: substrate
concentration, temperature and pH.
- The presence of inhibitors may also hinder substrates from binding to
enzymes which further hinders enzymatic action.
 TO DETERMINE THE EFFECT OF
CHANGES IN TEMPERATURE,
GOAL OF THE SUBSTRATE CONCENTRATION
AND PH ON AN ENZYME
EXPERIMENT (SPECIFICALLY SALIVARY
AMYLASE)
MATERIALS AND
METHODOLOGY
REAGENTS USED AND SOLUTIONS PREPARED
ENZYME SUBSTRATE IKI

Amylase was obtained DESCRIPTION -drop of iodine solution was

put in a spot plate
from a member of the ● 5mL starch
group, filtered and was ● 2mL NaCl -the iodine solution was
wrapped in foil because of its
then diluted to 1:10 ● 2mL phosphate
sensitivity to light
ratio using distilled buffer
● 37 deg C*
water.
CONTROL SETUP: GENERAL FLOW OF THE
EXPERIMENT
STEP 1 STEP 2 STEP 3

PREPARATION OF THE ADDITION OF THE DETERMINATION OF

SUBSTRATE ENZYME ACHROMIC POINT
When the solution was When the solution was a drop of the digestion mixture
was added to a drop of iodine
mixed, the test tube was already in the same
solution in a spot plate every 1
then immersed in a water temperature as the water minute
bath maintained at 37 ̊C. bath, 1ml of amylase was
ACHROMIC POINT: until no
added and the time of its
color change was observed
addition was recorded.
● IKI WAS ADDED TO
DETERMINE ACHROMIC
POINT
EXPERIMENTAL TEMPERATURE

SETUPS PH

ENZYME CONCENTRATION
CHANGES IN DIFFERENT
SUBSTRATE CONCENTRATION
VARIABLES
 10oC 1:10 1% 3
25oC 1:20 2% 4
37oC 1:40 3% 5
40-50oC 1:60 4% 6.8
60oC 1:80 5% 7
80oC 1:100 6% 8

ENZYME SUBSTRATE
TEMPERATURE pH
CONCENTRATION CONCENTRATION
CALCS
RESULTS AND
DISCUSSION
SALIVARY AMYLASE
● CATALYST INVOLVED IN FIRST STAGE OF STARCH METABOLISM

● HYDROLYSIS OF STARCH TO MALTOSE

● ENDOHYDROLASE: CLEAVES HYDRATED STARCH MOLECULES AT RANDOM

INTO 2 SMALLER MOLECULES

● CLEAVAGE OF ANY ACCESSIBLE (1,4) BOND

● ACTIVITY IS AFFECTED BY MULTIPLE FACTORS

EXPERIMENTAL SETUP
STARCH SOLUTION AT 37 DEGREES
HEATING → Solubilisation and
gelatinisation
COOLING → leads to recrystallization
of retrograded starch
RETROGRADED STARCH: resists
amylase activity
IKI INDICATOR
BLUE STARCH-IODINE COMPLEX
BLUE COLOR REACTION: reaction with
unbranched linear portions of starch
HYDROLYZED STARCH: less sites for
iodine to bind with, so lighter color
ACHROMIC POINT: light brown, no
trace of blue
EFFECT OF TEMPERATURE ON ENZYME ACTIVITY
OPTIMUM OPTIMUM
UNTIL 40 DEGREES ABOVE 40 DEGREES
TEMPERATURE TEMPERATURE

KINETIC ENERGY ENZYME INACTIVATION REACTION RATE AT

DENATURATION MAXIMUM
↑ Temperature
↑ denaturation Rate of enzyme
↑ kinetic energy of the
destruction BALANCED
substrate molecules Protein breakdown
by increase in
↑ reaction rate ↓ rate of reaction enzyme-substrate
reactivity
 Optimal temp: 37
DEGREES
EFFECT OF
TEMPERATURE
ON ENZYME
ACTIVITY
EFFECT OF PH ON ENZYME ACTIVITY
OPTIMUM
TEMPERATURE

3.8 6.9 9.4

INCREASE IN PH
DECREASE IN PH
↓ positive sites
↑ positive sites
EFFECT OF PH ON max activity

ENZYME
ACTIVITY
OPTIMUM PH AT 6.8
RATE OF REACTION
● NORMAL EQUATION: S → P

● EQUATION PROPOSED BY THE MICHAELIS-MENTEN EQUATION:

○ ASSUMPTION 1: NEGLECTS K4

○ ASSUMPTION 2: ES COMPLEX IS A STEADY STATE INTERMEDIATE

■ Rate of formation = Rate of Breakdown

■ NOTE: enzyme isn’t consumed after reaction

EFFECT OF ENZYME CONCENTRATION
UNLIMITED SUBSTRATE LIMITED SUBSTRATE (CONSTANT)
VS
SATURATION POINT DETERMINES
↑AMOUNT OF ENZYME
MAXIMUM RATE OF REACTION
↑ REACTION RATE Saturation point: all enzyme active
sites occupied by substrate
↑AMOUNT OF SUBSTRATE

↑ REACTION RATE UNTIL SATURATION POINT

 EFFECT OF
ENZYME
CONCENTRATION
ON ENZYME
ACTIVITY
EFFECT OF SUBSTRATE CONCENTRATION
UNLIMITED ENZYME LIMITED ENZYME (CONSTANT)
VS
SATURATION POINT DETERMINES
↑AMOUNT OF SUBSTRATE
MAXIMUM RATE OF REACTION
↑ REACTION RATE Saturation point: all enzyme active
sites occupied by substrate
↑AMOUNT OF SUBSTRATE

↑ REACTION RATE UNTIL SATURATION POINT

 EFFECT OF
SUBSTRATE
CONCENTRATION
ON ENZYME
ACTIVITY
 MICHAELIS-
MENTEN PLOT
LINEWEAVER-
BURKE PLOT

y = m x + b
y = 1E+17 x + 5E+19
SUMMARY
 THEORETICAL RESULTS ON ENZYME ACTIVITY
PARABOLIC:
EXPONENTIAL
decreasing activity at
DECREASE CURVE:
both ends from
Decreasing until
optimum temperature
plateau is reached

ENZYME SUBSTRATE
TEMPERATURE pH
CONCENTRATION CONCENTRATION

EXPONENTIAL PARABOLIC: peaks at

DECREASE CURVE: 6.8
Decreasing until
plateau is reached
EXPERIMENTAL RESULTS ON ENZYME ACTIVITY
EXPONENTIAL
DECREASE CURVE:
Decreasing until
plateau is reached

ENZYME SUBSTRATE
TEMPERATURE pH
CONCENTRATION CONCENTRATION

EXPONENTIAL PARABOLIC: peaks at

DECREASE CURVE: 6.8
decreasing until plateau
is reached
EXPERIMENTAL RESULTS ON ENZYME ACTIVITY
EXPONENTIAL
DECREASE CURVE:
Decreasing until
plateau is reached

ENZYME SUBSTRATE
TEMPERATURE pH
CONCENTRATION CONCENTRATION

PARABOLIC: peaks at
6.8
EXPERIMENTAL RESULTS ON ENZYME ACTIVITY
EXPONENTIAL
DECREASE CURVE:
Decreasing until
plateau is reached

ENZYME SUBSTRATE
TEMPERATURE pH
CONCENTRATION CONCENTRATION

PARABOLIC: peaks at
6.8
EXPERIMENTAL RESULTS ON ENZYME ACTIVITY
EXPONENTIAL
DECREASE CURVE:
Decreasing until
plateau is reached

ENZYME SUBSTRATE
TEMPERATURE pH
CONCENTRATION CONCENTRATION

GENERAL TREND OF
RESULTS IN ACTUAL
EXPERIMENT
THEORETICAL MICHAELIS-MENTEN PLOT

SUBSTRATE
CONCENTRATION
INVERSE EXPONENTIAL CURVE:
increasing until plateau is reached
EXPERIMENTAL MICHAELIS-MENTEN PLOT

SUBSTRATE
CONCENTRATION
INVERSE EXPONENTIAL CURVE:
increasing until plateau is reached
 RESULTS SHOWED TRENDS IN
CONCLUSION LINE WITH RELATED LITERATURE
AND THEORETICAL DATA