2. What Are Enzymes?What Are Enzymes?
Most enzymes are
ProteinsProteins ((tertiary
and quaternary
structures)
Act as CatalystCatalyst to
accelerates a
reaction
Not permanentlyNot permanently
changed in the
process
2
3. Classes of enzymes
1. Oxidoreductases = catalyze oxidation-
reduction reactions (NADH)
2. Transferases = catalyze transfer of functional
groups from one molecule to another.
3. Hydrolases = catalyze hydrolytic cleavage
4. Lyases = catalyze removal of a group from or
addition of a group to a double bond, or other
cleavages involving electron rearrangement.
5. Isomerases = catalyze intramolecular
rearrangement.
6. Ligases = catalyze reactions in which two
molecules are joined.
Enzymes named for the substrates and type of
reaction
4.
5. Co-enzymes
• Non-protein molecules that help enzymes
function
• Associate with active site of enzyme
• Enzyme + Co-enzyme = holoenzyme
• Enzyme alone = apoenzyme
• Organic co-enzymes – thiamin, riboflavin,
niacin, biotin
• Inorganic co-enzymes – Mg ++
, Fe++
, Zn++
,
Mn++
8. Initial Velocities
[S] = 1 mM
[S] = 5 mM
[S] = 10 mM∆[P]/∆T = Vo10 mM
D[P]/DT = Vo5 mM
D[P]/DT = Vo1 mM
[P]
time
9. Active SiteActive Site
A restricted regionrestricted region of an enzymeenzyme
molecule which bindsbinds to the
substratesubstrate.
9
Enzyme
Substrate
Active
Site
10. Induced FitInduced Fit
A change in
the shapeshape of an
enzyme’s active
site
InducedInduced by the
substrate
10
11. Induced FitInduced Fit
A changechange in the configurationconfiguration of an
enzyme’s activeenzyme’s active sitesite (H+ and ionic
bonds are involved).
InducedInduced by the substratesubstrate..
11
Enzyme
Active Site
substrate
induced fit
13. Understanding Vmax
The theoretical maximal velocity
• Vmax is a constant
• Vmaxis the theoretical maximal rate of the reaction -
but it is NEVER achieved in reality
• To reach Vmax would require that ALL enzyme
molecules are tightly bound with substrate
• Vmax is an approached as substrate is increased
14. Understanding Km
The "kinetic activator constant"
• Km is a constant
• Km is a constant derived from rate constants
• Km is, under true Michaelis-Menten conditions, an
estimate of the dissociation constant of E from S
• Small Km means tight binding; high Km means
weak binding
15. Km = [S] @ ½ Vmax
(units moles/L=M)
(1/2 of enzyme bound to S)
Vmax = velocity where all of the
enzyme is bound to substrate
(enzyme is saturated with S)
16. What does kcat mean?
1. kcat is the 1st
order rate constant describing ES
E+P
2. Also known as the turnover # because it describes
the number of rxns a molecule of enzyme can
catalyze per second under optimal condition.
3. Most enzyme have kcat values between 102
and 103
s-1
4. For simple reactions k2 = kcat , for multistep rxns kcat
= rate limiting step
E + S ES E + P
k1
k-1
kcat
17. What does kcat/Km mean?
• It measures how the enzyme
performs when S is low
• kcat/Km describes an enzymes
preference for different
substrates = specificity constant
• The upper limit for kcat/Km is the
diffusion limit - the rate at
which E and S diffuse together
(108
to 109
m-1
s-1
)
• Catalytic perfection when kcat/Km =
diffusion rate
• More physiological than kcat
18. Limitations of M-M
1. Some enzyme catalyzed rxns show more complex behavior
E + S<->ES<->EZ<->EP<-> E + P
With M-M can look only at rate limiting step
2. Often more than one substrate
E+S1<->ES1+S2<->ES1S2<->EP1P2<-> EP2+P1<-> E+P2 Must
optimize one substrate then calculate kinetic parameters
for the other
3. Assumes k4 = 0
4. Assume steady state conditions
19. How do you get values for Vmax, Km and kcat?
• Can determine Km and Vmax experimentally
• Km can be determined without an absolutely
pure enzyme
• Kcat values can be determined if Vmax is known and
the absolute concentration of enzyme is known
(Vmax = kcat[Etotal]
24. Ping-Pong Reactions
E (EA)(FP) (F) (FB)(EQ) E
A BP Q
•In Ping-Pong rxns first product released before
second substrate binds
•When E binds A, E changes to F
•When F binds B, F changes back to E
25. Enzyme Inhibition
• Inhibitor – substance that binds to an enzyme and
interferes with its activity
• Can prevent formation of ES complex or prevent ES
breakdown to E + P.
• Irreversible, Reversible and Allosteric Inhibition
• Irreversible inhibitor binds to enzyme through covalent
bonds (binds irreversibly)
• Reversible Inhibitors bind through non-covalent interactions
(disassociates from enzyme)
26. 1. Irreversible Inhibitors:
Covalently bind and inactive enzyme
Suicide Inhibitor
2. Reversible Inhibitors:
E + S <-> ES -> E + P
E + I <-> EI
Ki = [E][I]/[EI]
• Competitive
• Uncompetitive
• Non-competitive
28. 3. Allosteric Inhibitor
Allosteric Modulator/effectors
Activator site
Inhibitor site
Classes of allosteric enzyme
1.K-class of allosteric enzyme: km changes
and not V max
2.V-class of allosteric enzyme alter V max not km
29. Data from a single experiment
performed with at a single [S].
(single point on Vo vs. [S] plot)
30. Regulation of Enzyme Activity
Enzyme quantity – regulation of gene expression (Response time =
minutes to hours)
a) Transcription
b) Translation
c) Enzyme turnover
Enzyme activity (rapid response time = fraction of seconds)
a) Allosteric regulation
b) Covalent modification
c) Association-disassociation’
d) Proteolytic cleavage of proenzyme
31. 31
Enzyme Used in beverage Manufacturing-
Enzymes are, widely used beverage and brewing industries to
achieve specific objectives:
• Fermentation
• Clarification
• Pectin manufacturing- pecteolytic enzymes
• Brewing industry
• Production of syrup- Takadiastase
• Coffee bean fermentation- pectinase
• High test Mollases- Invertase
• Beer manufacturing-mashing- amylase
• Production of cheese, beer, spirits.
32. 32
Treatment of fruit pulp with pecteolytic enzyme mixture gives the
following benefits:
(i) Elimination of juice/wine cloudiness,
(ii) reduced solution viscosity,
(iii) Increased juice yields, e.g., a 15% increase in
case of white grapes, and
(iv) Shorter fermentation period in case of wine
making. In addition,
(v) pectins stabilize the cell debris in a colloidal
state.
(vi)binding constituents are converting from
polyform to oligoform which is useful for human body.
Application of enzymes in fruit juice manufacturing
