Enzyme - good lecture

1. Enzymes Are the Agents ofEnzymes Are the Agents of
Metabolic FunctionMetabolic Function
• Acting in sequence, enzymes form metabolic
pathways by which:
– nutrient molecules are degraded,
– energy is released and converted into metabolically
useful forms,
– precursors are generated and transformed to create
thousands of distinctive biomolecules found in any
living cell
(Figure 14.2).
• Situated at key junctions of metabolic pathways
are specialized regulatory enzymes capable of:
– sensing the momentary metabolic needs of the cell
– adjusting their catalytic rates accordingly.

2. Figure 14.2
• The breakdown
of glucose by
glycolysis
provides a prime
example of a
metabolic
pathway.
• 10 enzymes
mediate the
reactions of
glycolysis.
• Enzyme 4,
fructose 1,6,
biphosphate
aldolase,
catalyzes the C-C
bond- breaking
reaction in this
pathway

3. 1 · Enzymes—Catalytic Power,1 · Enzymes—Catalytic Power,
Specificity, and RegulationSpecificity, and Regulation
Enzymes are characterized by 3
distinctive features:
• catalytic power,
• specificity,
• Regulation.

4. Catalytic Power
• Enzymes display enormous catalytic
power, accelerating reaction rates as
much as 1016
over uncatalyzed levels,
(far greater than any synthetic catalysts
can achieve).
• Enzymes accomplish these astounding
feats:
– in dilute aqueous solutions
– under mild conditions of temperature and pH.

5. For exampleFor example, the enzyme jack bean, the enzyme jack bean ureaseurease
catalyzes the hydrolysis of urea:catalyzes the hydrolysis of urea:
• At 20°CAt 20°C:
– the rate constant for the enzyme-catalyzed reaction
is 3.104
/sec;
– the rate constant for the uncatalyzed hydrolysis of
urea is 3.10-10
/sec.
• Thus, the ratio of the catalyzed rate to theThus, the ratio of the catalyzed rate to the
uncatalyzed rate of reaction is:uncatalyzed rate of reaction is:
– 10101414
-- defined as the relativedefined as the relative catalytic powercatalytic power of anof an
enzyme,enzyme, soso
• thethe catalytic power of urease is 10catalytic power of urease is 101414
..

6. SpecificitySpecificity
• Enzymes selectively recognize proper
substrates over other molecules
• Enzymes produce products in very high yields
- often much greater than 95%
• Specificity is controlled by structure - -
-the unique fit of substrate with enzyme
controls the selectivity for substrate and
the product yield

7. • A given enzyme is very selective, both in:
– the substances with which it interacts (substrates)
– the reaction that it catalyzes.
This selective qualities is the Specificity of an enzyme
• In an enzyme-catalyzed reaction:
– no wasteful by-products are produced
(can be contrasted with experiences in the organic
chemistry lab (yields of 50% or even 30%).
– the products are also very specific.

8. • Interaction between an enzyme and its substrates
occurs through molecular recognition based on
structural complementarity - the basis of specificity.
• The active site: specific site on the enzyme where
substrate binds and catalysis occurs.

10. Lineweaver-Burk (doubleLineweaver-Burk (double
reciprocal plot)reciprocal plot)
• If the reciprocal (1/X) of the Michaelis-Menten
equation is done, after algebraic simplification
the following equation results:
• This relation is written in the format of the
equation for a straight line, y = mx + b, where
y = 1/vo, m (slope) = Km/Vmax, x = 1/[S] and the y-
intercept, b = 1/Vmax. When this relation is
plotted,the result is a straight line graph

11. Lineweaver-Burk (doubleLineweaver-Burk (double
reciprocal plot) (cont)reciprocal plot) (cont)

12. Uses of double reciprocalUses of double reciprocal
plotplot
• The x intercept value is equal to -1/Km.
The biggest advantage to using the
double reciprocal plot is a more accurate
determination of Vmax, and hence Km. It is
also useful in characterizing the effects of
enzyme inhibitors and distinguishing
between different enzyme mechanisms.

13. Enzyme Inhibitor TypesEnzyme Inhibitor Types
• Inhibitors of enzymes are generally molecules
which resemble or mimic a particular enzymes
substrate(s). Therefore, it is not surprising that
many therapeutic drugs are some type of
enzyme inhibitor. The modes and types of
inhibitors have been classified by their kinetic
activities and sites of actions. These include
Reversible Competitive Inhibitors,
Reversible Non-Competitive Inhibitors,
and Irreversible Inhibitors

14. Competitive InhibitionCompetitive Inhibition
Vmax - No change
Km INCREASES - indicates a direct interaction
of the inhibitor in the active site

15. • Competitive inhibitors compete with the substrate
for binding at the active site (as E + I). In the double
reciprocal plot for a competitive inhibitor acting at
the substrate site for the following reasons, notice
with increasing concentration of inhibitor, the Vmax
does not change; however, the Km of the
substrate is increased. This also reflects the
reversible nature of the inhibitor; there is always
some concentration of substrate which can displace
the inhibitor.
Reversible CompetitiveReversible Competitive
InhibitionInhibition

16. Non-Competitive InhibitionNon-Competitive Inhibition
Vmax DECREASES - inhibitor affects rate of reaction
by binding to site other than substrate active-site
Km - No change

17. • Non-competitive inhibitors combine with both the enzyme
(E + I) and the enzyme-substrate (EI + S) complex. The
inhibitor binds to a site other that the substrate site, and is
thus independent of the presence or absence of
substrate. This action results in a conformational change
in the protein that affects a catalytic step and hence
decreases or eliminates enzyme activity (formation of P).
Notice in the reciprocal plot, a non-competitive inhibitor
does not affect the binding of the substrate (Km), but it
does result in a decrease in Vmax. This can be explained
by the fact that since inhibitor bound to an enzyme
inactivates it, the more EI formed will lower [ES] and thus
lower the overall rate of the reaction Vmax.
Reversible Non-Competitive InhibitionReversible Non-Competitive Inhibition

18. • Irreversible inhibitors generally result in the destruction or
modification of an essential amino acid required for enzyme
activity. Frequently, this is due to some type of covalent link
between enzyme and inhibitor. These types of inhibitors
range from fairly simple, broadly reacting chemical modifying
reagents (like iodoacetamide that reacts with cysteines) to
complex inhibitors that interact specifically and irreversibly
with active site amino acids. (termed suicide inhibitors).
These inhibitors are designed to mimic the natural substrate
in recognition and binding to an enzyme active site. Upon
binding and some catalytic modification, a highly reactive
inhibitor product is formed that binds irreversibly and
inactivates the enzyme. Use of suicide inhibitors have proven
to be very clinically effective
Irreversible InhibitorsIrreversible Inhibitors

19. Irreversible Inhibitor: AllopurinolIrreversible Inhibitor: Allopurinol

21. • Effect of pH on Enzymatic Activity
Enzyme-substrate recognition and the catalytic events
that ensue are greatly dependent on pH.
An enzyme possesses an array of ionizable side chains
and prosthetic groups that:
– determine its secondary and tertiary structure
– may be intimately involved in its active sit
The substrate itself often has ionizing groups, and one
or another of the ionic forms may preferentially interact
with the enzyme.

22. Enzymes in general are active only over a limited
pH range and most have a particular pH at which
their catalytic activity is optimal.
• These effects of pH may be due to effects on Km or
Vmax or both.
• Figure 14.11 illustrates the relative activity of 4
enzymes as a function of pH.
• Although the pH optimum of an enzyme often
reflects the pH of its normal environment, the
optimum may not be precisely the same.
• This difference suggests that the pH-activity
response of an enzyme may be a factor in the
intracellular regulation of its activity.

23. Fig.
The pH activity
profiles of four
different enzymes
Trypsin, an intestinal
protease, has a slightly
alkaline pH optimum.
Pepsin, a gastric
protease, acts in the
acidic confines of the
stomach and has a pH
optimum near 2.
Papain, a protease
found in papaya, is
relatively insensitive to
pHs between 4 & 8.
Cholinesterase
activity is pH-sensitive
below pH 7 but not
between pH 7 and 10.

24. Effect of Temperature on
Enzymatic Activity
• Like most chemical reactions, the rates of enzyme-
catalyzed reactions generally increase with
increasing temperature.
• However, at temperatures above 50° to 60°C,
enzymes typically show a decline in activity (Fig).
• Two effects are operating here:
– (a) the characteristic increase in reaction rate
with temperature, and
– (b) thermal denaturation of protein structure
at higher temperatures.

25. • (Most enzymatic reactions double in rate for
every 10°C rise in temperature (that is, Q10 =
2, where Q10 is defined as the ratio of
activities at two temperatures 10° apart) as
long as the enzyme is stable and fully active).
• Some enzymes, those catalyzing reactions
having very high activation energies, show
proportionally greater Q10 values.

26. • The increasing rate with increasing
temperature is ultimately offset by the
instability of higher orders of protein
structure at elevated temperatures, where the
enzyme is inactivated.
• Not all enzymes are quite so thermally labile.
• For example, the enzymes of thermophilic
bacteria (thermophilic = ”heat-loving”) found
in geothermal springs retain full activity at
temperatures in excess of 85°C.

29. COENZYMESCOENZYMES
- Some enzymes require an additional
component for activity
- These components are called
COENZYMES
• Used at the active site of the
enzyme
• Not covalently bound to the
enzyme
• Can be small organic molecules
or metal ions
• Many are structurally related to
vitamins
• They are regenerated for further
reactions
- PROSTHETIC GROUPS are
COENZYMES that ARE covalently
bound to an enzyme and therefore are
always present

30. • Coenzymes:
– are usually actively involved in the catalytic
reaction of the enzyme,
– often serving as intermediate carriers of functional
groups in the conversion of substrates to products.
• In most cases, a coenzyme is firmly associated with
its enzyme (even by covalent bonds) and is referred
to as prosthetic groups of the enzyme.
• Holoenzyme: the catalytically active complex of
protein and prosthetic group.
• Apoenzyme: the protein without the prosthetic
group; it is catalytically inactive

34. REGULATION OF ENZYMEREGULATION OF ENZYME
ACTIVITYACTIVITY

35. Regulation
• Regulation of enzyme activity is achieved in:
– controls over the amount of enzyme protein
produced by the cell
– more rapid,reversible interactions of the enzyme
with metabolic inhibitors and activators.
• Because most enzymes are proteins, the functions of
enzymes are due to the remarkable versatility
found in protein structures.

36. - Regulation means to make an enzyme more or less active
- In general, regulation is necessary to control the rates of
reactions and to properly synchronize all of the metabolic
reactions in the cell. Keep it running like a finely tuned
machine.
How can you change enzyme behavior in a cell?

37. - Induction/repression
• Change the rate of enzyme synthesis and/or degradation of
the enzyme
• Change cellular distribution of the enzyme
- Modify the intrinsic properties of the enzyme
• Non-covalent interactions
1. Bind regulatory molecules reversibly (e.g. proteins,
lipids, small molecules)
• Reversible Covalent Modifications
1. Phosphorylation of serine, threonine or tyrosine
2. Methylation of glutamate residues
o Used in bacteria as food sensor

40. • Irreversible Covalent Modifications
1. Isoprenylation, acylation, palmitoylation – addition of fatty acids
and fatty acid derivatives
2. Glycosylation – addition of sugars to Asparagine
3. Proteolytic cleavage
o ZYMOGENS – inactive precursor to an enzyme; activated by
cleavage of a specific peptide bond
o Why would this be useful? Let’s look at examples:
§ Proteolytic enzymes TRYPSIN and
CHYMOTRYPSIN
§ Initially synthesized as trypsinogen and chymo-
trypsinogen which are both inactive
§ Formed in the pancreas where they would do damage if
active
§ In the small intestine, where their digestive properties
are needed, they are ACTIVATED by cleavage of
specific peptide bonds.

41. ZYMOGENS ARE INACTIVE UNTIL REACH PROPER ENVIRONMENT!!
CHYMOTRYPSINOGEN:
INACTIVE PRECURSOR OF CHYMOTRYPSIN

43. General Properties:General Properties:
Regulatory EnzymesRegulatory Enzymes
• The biochemical pathways are composed of
groups of coordinated enzymes that perform a
specific metabolic process.
• In general, these enzyme groups are composed of
many enzymes, only a few of which are regulated
by the mechanisms described in this lecture.
• Regulatory enzymes are usually the enzymes
that are the rate-limiting, or committed step, in a
pathway, meaning that after this step a particular
reaction pathway will go to completion.

44. General Properties:General Properties:
Regulatory Enzymes (cont)Regulatory Enzymes (cont)
• Regulatory enzymes are at or near the initial
steps in a pathway, or part of a branch point or
cross-over point between pathways (where a
metabolite can be potentially converted into
several products in different pathways).
• A cell needs to conserve energy - therefore
costly (in metabolic terms) biosynthetic reaction
pathways will not be operational unless a
particular metabolite is required at a given time.

45. General Properties:General Properties:
Regulatory Enzymes (cont)Regulatory Enzymes (cont)
• When acting as catalysts, enzyme mediated-
reactions should be reversible.
• However, regulatory enzymes frequently catalyze
thermodynamically irreversible reactions, that is, a
large negative free energy change (-∆G) greatly
favors formation of a given metabolic product
rather than the reverse reaction.
• Thus, regulation of enzyme activity, usually at the
committed step of the pathway, is critical for
supplying and maintaining cellular metabolitic and
energy homeostasis.

46. Two General Mechanisms thatTwo General Mechanisms that
Affect Enzyme Activity:Affect Enzyme Activity:
• 1) control of the overall quantities
of enzyme or concentration of
substrates present
• 2) alteration of the catalytic
efficiency of the enzyme

48. Regulation of EnzymeRegulation of Enzyme
ConcentrationsConcentrations
• The overall synthesis and degradation of a
particular enzyme, also termed its
turnover number, is one way of
regulating the quantity of an enzyme. The
amount of an enzyme in a cell can be
increased by increasing its rate of
synthesis, decreasing the rate of its
degradation, or both.

49. • Induction (an increase caused by an effector
molecule) of enzyme synthesis is a common
mechanism - this can manifest itself at the level of
gene expression, RNA translation, and post-
translational modifications.
• The actions of many hormones and/or growth
factors on cells will ultimately lead to an increase in
the expression and translation of "new" enzymes
not present prior to the signal.
Regulation of EnzymeRegulation of Enzyme
Concentrations: InductionConcentrations: Induction

50. Regulation of EnzymeRegulation of Enzyme
Concentrations: DegradationConcentrations: Degradation
• The degradation of proteins is constantly
occuring in the cell, yet the molecular
mechanisms that determine when and which
enzymes will be degraded are poorly
understood.
• The turnover number of an enzyme can be
used for general comparison with other
enzymes or other enzyme systems, yet these
numbers can vary from minutes to hours to
days for different enzymes.

51. • Protein degradation by proteases is
compartmentalized in the cell in the lysosome
(which is generally non-specific), or in
macromolecular complexes termed proteasomes
• Degradation by proteasomes is regulated by a
complex pathway involving transfer of a 76 a.a
polypeptide, ubiquitin, to targeted proteins.
• Ubiquination of protein targets it for degradation
by the proteasome.
This pathway is highly conserved in
eukaryotes, but still poorly understood
Regulation of EnzymeRegulation of Enzyme
Concentrations: Degradation (cont)Concentrations: Degradation (cont)

52. Regulation of EnzymeRegulation of Enzyme
Concentrations: Degradation (cont)Concentrations: Degradation (cont)
• Proteolytic degradation is an irreversible
mechanism.
For examples, rapid proteolytic degradation of
enzymes that were activated in response to some
stimulus (e.g, in a signal transduction response).
This type of down-regulation allows for a transient
response to a stimulus instead of a continual
response.
Establishing the links between proteasomes,
ubiquination and signal transduction pathways is
currently a very active research area

53. Zymogens: InactiveZymogens: Inactive
Precursor ProteinsPrecursor Proteins
• A clinically important mechanism of controlling
enzyme activity is the case of protease enzymes
involved (predominantly) in food digestion and
blood clotting.
• Protease enzymes (enzymes that degrade
proteins) like pepsin, trypsin and chymotrypsin
are synthesized first as larger, inactive precursor
proteins termed zymogens (specifically
pepsinogen, trypsinogen, and
chymotrypsinogen, respectively).

54. Zymogen Protease ExamplesZymogen Protease Examples
Chymotrypsinogen
cleavage sites to
yield active
chymotrypsin

55. Zymogens (cont)Zymogens (cont)
• Activation of zymogens by proteolytic cleavage
result in irreversible activation.
• Zymogen forms allow proteins to be transported or
stored in inactive forms that can be readily
converted to active forms in response to some type
of cellular signal.
• Thus they represent a mechanism whereby the
levels of an enzyme/protein can be rapidly
increased (post-translationally).
• Other examples of zymogens include proinsulin,
procollagen and many blood clotting enzymes.

56. Allosteric EnzymesAllosteric Enzymes
• Allosteric enzymes - from the Greek allos for
"other" and stereos for "shape" (or site) meaning
"other site".
• These enzymes function through reversible, non-
covalent binding of a regulatory metabolite at a site
other than the catalytic, active site.
• When bound, these metabolites do not participate in
catalysis directly, but lead to conformational
changes in one part of an enzyme that then affect
the overall conformation of the active site (causing
an increase or decrease in activity, hence these
metabolites are termed allosteric activators or

57. Allosteric ExampleAllosteric Example
• Feedback Inhibition - This occurs when an
end-product of a pathway accumulates as the
metabolic demand for it declines.
• This end-product in turn binds to the regulatory
enzyme at the start of the pathway and
decreases its activity - the greater the end-
product levels the greater the inhibition of
enzyme activity.
• This can either effect the Km or Vmax of the
enzyme reaction.

58. Metabolic Pathway Product/Metabolic Pathway Product/
Feedback InhibitionFeedback Inhibition

59. Regulation by ModulatorRegulation by Modulator
Proteins - CalmodulinProteins - Calmodulin
Calmodulin is a small protein
(17 kDa) that can bind up to four
calcium ions (blue dots) in the
two globular domains. When
calciumis bound, calmodulin acts
as a protein co-factor to stimulate
the activity of target regulatory
kinases like phosphorylase kinase,
myosin kinase, Ca-ATPase and a
Ca/calmodulin-dependent
protein kinase. It is the structural
conformation of Ca-calmodulin
that makes it an active co-factor

60. Regulation of Enzyme Activity byRegulation of Enzyme Activity by
Covalent ModificationsCovalent Modifications
• Another common regulatory mechanism is the
reversible covalent modification of an enzyme.
• Phosphorylation, whereby a phosphate is
transferred from an activated donor (usually ATP)
to an amino acid on the regulatory enyme, is the
most common example of this type of regulation.
• Frequently this phosphorylation occurs in response
to some stimulus (like a hormone or growth factor)
that will either activate or inactivate target enzymes
via changes in Km or kcat.

61. Phosphorylation/Signal TransductionPhosphorylation/Signal Transduction
• Phosphorylation of one enzyme can lead to
phosphorylation of a different enzyme which in turn
acts on another enzyme, and so on.
• An example of this type of phosphorylation cascade
is the response of a cell to cyclic AMP and its effect
on glycogen metabolism.
• Use of a phosphorylation cascade allows a cell to
respond to a signal at the cell surface and transmit
the effects of that signal to intracellular enzymes
(usually within the cytosol and nucleus) that modify a
cellular process.
• This process is generically referred to as being part of
a signal transduction mechanism

62. Signaling Regulation of GlycogenSignaling Regulation of Glycogen
Synthase and PhosphorylaseSynthase and Phosphorylase
A-forms, most active B-forms, less active

63. Other covalent modificiations:Other covalent modificiations:
• Prenylation, Myristoylation, Palmitoylation:
• The covalent addition of hydrophobic, acyl
fatty acid or isoprenoid groups to soluble
proteins/enzymes can alter their intracellular
location.
• This type of hydrophobic acylation generally
causes target proteins to associate with a
membrane rather than the cytosol.
• Thus, it represents a mechanistic and
functional re-compartmentalization of the
target protein/enzyme

64. Allosteric and PhosphorylationAllosteric and Phosphorylation
Regulation - Glycogen PhosphorylaseRegulation - Glycogen Phosphorylase

67. Enzyme Nomenclature
• Traditionally, enzymes often were named by adding
the suffix-ase to the name of the substrate upon
which they acted, such as:
– Urease for the urea-hydrolyzing enzyme, or
– Phosphatase for enzymes hydrolyzing phosphoryl
groups from organic phosphate compounds).
• Other enzymes acquired names bearing little
resemblance to their activity, such as:
– Catalase: the peroxide-decomposing enzyme,
– Trypsin and pepsin: the proteolytic enzymes (proteases) of
the digestive tract.

68. • International Commission on Enzymes (1956) created
a systematic basis for enzyme nomenclature
• All enzymes now are classified and formally named
according to the reaction they catalyze
(common names for many enzymes remain in use).
• Six classes of reactions are recognized

69. • Within each class are subclasses,
• Under each subclass are sub-subclasses
• Within sub-subclasses are individual enzymes.
• Classes (1), subclasses (2), sub-subclasses (3),
individual entries (4) are each numbered
(so that a series of 4 numbers serves to specify
a particular enzyme).
• A systematic name, descriptive of the reaction,
is also assigned to each entry.

70. Table 14.1
Systematic Classification of Enzymes According to
the Enzyme Commission
E.C. Number Systematic Name and Subclasses
1
Oxidoreductases
(oxidation–reduction reactions)
1.1 Acting on CH—OH group of donors
1.1.1 With NAD or NADP as acceptor
1.1.3 With O2 as acceptor
1.2 Acting on the group of donors
1.2.3 With O2 as acceptor
1.3 Acting on the CH—CH group of donors
1.3.1 With NAD or NADP as acceptor

71. 2 Transferases (transfer of functional groups)
2.1 Transferring C-1 groups
2.1.1 Methyltransferases
2.1.2
Hydroxymethyltransferases and
formyltransferases
2.1.3
Carboxyltransferases and
carbamoyltransferases
2.2 Transferring aldehydic or ketonic residues
2.3 Acyltransferases
2.4 Glycosyltransferases
2.6 Transferring N-containing groups
2.6.1 Aminotransferases
2.7 Transferring P-containing groups
2.7.1 With an alcohol group as acceptor

72. 3 Hydrolases (hydrolysis reactions)
3.1 Cleaving ester linkage
3.1.1 Carboxylic ester hydrolases
3.1.3 Phosphoric monoester hydrolases
3.1.4 Phosphoric diester hydrolases
4 Lyases (addition to double bonds)
4.1 C=C lyases
4.1.1 Carboxy lyases
4.1.2 Aldehyde lyases
4.2 C=O lyases
4.2.1 Hydrolases
4.3 C=N lyases
4.3.1 Ammoniα-lyases

73. 5 Isomerases (isomerization reactions)
5.1 Racemases and epimerases
5.1.3 Acting on carbohydrates
5.2 Cis-trans isomerases
6
Ligases
(formation of bonds with ATP cleavage)
6.1 Forming C—O bonds
6.1.1 Amino acid–RNA ligases
6.2 Forming C—S bonds
6.3 Forming C—N bonds
6.4 Forming C—C bonds
6.4.1 Carboxylases

74. To illustrate, consider the enzyme that catalyzes this
reaction:
ATP + D-glucose → ADP + D-glucose-6-phosphate
• A phosphate group is transferred from ATP to the C-6-
OH group of glucose, so the enzyme is a transferase
(Class 2, Table 14.1).
• Subclass 7 of transferases is enzymes transferring
phosphorus-containing groups, and sub-subclass 1
covers those phosphotransferases with an alcohol
group as an acceptor.
• Entry 2 in this sub-subclass is ATP: D-glucose-6-
phosphotransferase, its classification No
is 2.7.1.2.
• In use, this No
is written preceded by the letters E.C.
(denoting the Enzyme Commission): E.C. 2.7.1.2.

77. 6- Ribozymes and Abzymes6- Ribozymes and Abzymes
Relatively new discoveries
• Ribozymes - segments of RNA that display enzyme
activity in the absence of protein
– Examples: RNase P and peptidyl transferase
• Abzymes - antibodies raised to bind the transition
state of a reaction of interest