Enzymes are mostly proteins that act as catalysts to accelerate biochemical reactions without being permanently altered. They are classified based on the type of reaction they catalyze. Co-enzymes are non-protein molecules that help enzymes function by associating with their active sites. The Michaelis-Menten equation describes the relationship between substrate concentration and reaction rate. Kinetic parameters like Km, Vmax, and kcat can be determined experimentally to characterize enzyme kinetics. Enzymes are widely used in industries like food and beverage manufacturing for processes like fermentation, clarification, and mashing.

1. Enzymes

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

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++

6. E + S ES E + P
k1
k-1
k2
k-2
E S+ E S E + P

7. Plot Vo vs. [S]
Vo 1 mM
Vo 5 mM
Vo 10 mM

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

12. • Michaelis-Menton Equation
• Describes rectangular hyperbolic plot
• Vo = Vmax [S]
Km + [S]

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]

20. Obtaining Km, Vmax
Lineweaver-Burk plot

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

27. Types of Reversible Enzyme
Inhibitors

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

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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.

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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

33. 33

34. 34
Enzyme Production