Enzymes classification, factors affectin their activities

1. By Dr. M.H Abdelzaher
Enzymes

2. • Definitions:
• Enzymes are protein catalysts that increase the rate of reactions
without being changed in the overall process.
• Main function: Enzymes are biological catalysts responsible for
supporting almost all of the chemical reactions that maintain animal
homeostasis.
• Structure : The macromolecular components of almost all enzymes are
composed of protein, except for a class of RNA modifying catalysts
known as ribozymes. Ribozymes are molecules of ribonucleic acid that
catalyze reactions on the phosphodiester bond of other RNAs.

3. • Locations: Enzymes are found in all tissues and fluids
of the body.
• Intracellular enzymes catalyze the reactions of
metabolic pathways.
• Plasma membrane enzymes regulate catalysis within
cells in response to extracellular signals.
• Enzymes of the circulatory system are responsible for
regulating the clotting of blood. Almost every
significant life process is dependent on enzyme
activity.

4. Cofactors
• Some enzymes associate with a nonprotein
cofactor that is needed for enzymic activity.
• **Metal ions such as Fe , Zn.
• **Organic molecules, known as coenzymes,
that are often derivatives of vitamins. For
example, the coenzyme NAD contains niacin,
FAD contains riboflavin, and coenzyme A
contains pantothenic acid.

5. • Holoenzyme: refers to the enzyme with its cofactor.
• Apoenzyme:refers to the protein portion of the
holoenzyme.
• A prosthetic group: is a tightly bound coenzyme that
does not dissociate from the enzyme (for example,
the biotin bound to carboxylases)
• Zymogen : Some enzymes are synthesized as inactive
forms then proteolytic cleavage leads to generation
of active form like pepsinogen and pepsin.

7. Importance and function of cofactors or
coenzymes
• In the absence of the appropriate cofactor, the apoenzyme typically does
not show biologic activity (e.g. vitamin deficiency)
• The functional role of coenzymes is to act as transporters of chemical
groups from one reactant to another. The chemical groups carried can be as
simple as the hydride ion (H+ + 2e-) carried by NAD or the mole of hydrogen
carried by FAD; or they can be even more complex like the amine (-NH2)
carried by pyridoxal phosphate.
• Since coenzymes are chemically changed as a consequence of enzyme
action, it is often useful to consider coenzymes to be a special class of
substrates, or second substrates, which are common to many different
holoenzymes. In all cases, the coenzymes donate the carried chemical group
to an acceptor molecule and are thus regenerated to their original form.

10. Enzyme classification

11. Currently enzymes are grouped into six functional classes by
the International Union of Biochemists (I.U.B.).

12. Characteristics of enzymes
• 1) biological catalysts
• 2) not consumed during a chemical reaction
• 3) speed up reactions from 1000 - 1017
, times
• 4) exhibit stereospecificity --> act on a single stereoisomer
of a substrate
• 5) exhibit reaction specificity --> to group(pepsin/peptide
bond ) or similar compounds(lipase/TG) or even single
substrate (urease/ urea).
• 6)They are proteins (except ribozyme) and act within a
range of pH and temperature .

13. Nomenclature
• Typically add “-ase” to name of substrate e.g. lactase breaks down
lactose (dissacharide of glucose and galactose) or trivial name
(pepsin) or type of reaction (aminotransferases) .
• IUB classifies enzymes based upon the class mentioned before and
the group up on which enzyme acts and the type of coenzyme and
substrate . all of these give 4 numbers for each enzyme. (For
example, lactate dehydrogenase has the EC number 1.1.1.27
• (class 1, oxidoreductases; subclass 1.1, CH–OH group as electron
donor; sub-subclass 1.1.1, NAD as electron acceptor and 27 for
lactate).

14. Mechanisms of enzyme action
• **Transition state
• All chemical reactions have an energy barrier separating the reactants and the
products. This barrier, called the free energy of activation, is the energy
difference between that of the reactants and a high-energy intermediate that
occurs during the formation of product.
• For molecules to react, they must contain sufficient energy to overcome the
energy barrier i.e. reach the transition state at which the reactants are
energized and have high probability to make or break chemical bond leading to
formation of product. So the transition state is unstable intermediate form with
a structure between that of reactant and product.
• In the absence of an enzyme, only a small proportion of a population of
molecules may possess enough energy to reach the transition state between
reactant and product.

17. • **Catalysis by Proximity
• For molecules to react, they must come within bond forming distance of one
another. The higher their concentration, the more frequently they will
encounter one another and the greater will be the rate of their reaction.
• ** Acid-base catalysis
• Enzymes that use this have amino acid side chains that can donate or accept
protons to substrate. This can accelerate a chemical reaction by a factor of 10-
100 times.
• ** Catalysis by Strain (effort)
• Enzymes that catalyze lytic reactions which involve breaking a covalent bond
typically bind their substrates in a conformation slightly unfavorable for the
bond that will undergo cleavage. The resulting strain stretches or distorts the
targeted bond, weakening it and making it more vulnerable to cleavage.

19. • ** Covalent Catalysis
• Substrate forms a covalent bond with enzymes, and then part of
substrate is transferred to a second substrate in a 2 step process.
• A-X + E → X-E + A
• X-E + B → B-X + E
• One of the best-known examples of this mechanism is that
involving proteolysis by serine proteases, which include both
digestive enzymes (trypsin, chymotrypsin, and elastase) and
several enzymes of the blood clotting cascade. These proteases
contain an active site serine whose R group hydroxyl forms a
covalent bond with a carbonyl carbon of a peptide bond, thereby
causing hydrolysis of the peptide bond.

21. • **Substrates induce conformational changes in enzymes.
• Early in the last century, Emil Fischer compared the highly
specific fit between enzymes and their substrates to that of a
lock and its key and develops the lock and key theory but this
theory failed to account for the dynamic changes that
accompany catalysis so, the induced fit theory was introduced.
• The theory suggested that when substrates approach and bind
to an enzyme they induce a conformational change, a change
analogous to placing a hand (substrate) into a glove (enzyme).

22. Induced fit theory

23. • Enzyme binding sites. An enzyme binds the
substrates of the reaction and converts them to
products. The substrates are bound to specific
substrate binding sites on the enzyme through
interactions with the amino acid residues of the
enzyme.
• The spatial geometry required for all the
interactions between the substrate and the
enzyme makes each enzyme selective for its
substrates and ensures that only specific
products are formed

24. • Active catalytic site. The substrate binding
sites overlap in the active catalytic site of the
enzyme, the region of the enzyme where the
reaction occurs.
• Within the catalytic site, functional groups
provided by coenzymes, tightly bound
metals, and, of course, amino acid residues of
the enzyme, participate in catalysis.

26. Enzyme Kinetics
• Definition: Enzyme kinetics is the field of
biochemistry concerned with the quantitative
measurement of the rates of enzyme-catalyzed
reactions and the systematic study of factors
that affect these rates.

27. Initial velocity
• Initial velocity: Only initial reaction velocities Vi are used in the
analysis of enzyme reactions. This means that the rate of the reaction
is measured as soon as enzyme and substrate are mixed. At that time,
the concentration of product is very small and, therefore, the rate of
the back reaction from P to S can be ignored. Under these
conditions, only traces of product accumulate, hence the rate of the
reverse reaction is negligible.
• The initial velocity (vi ) of the reaction thus is essentially that of the
rate of the forward reaction. Under these conditions, vi is
proportionate to the concentration of enzyme. Measuring the initial
velocity therefore permits one to estimate the quantity of enzyme
present in a biologic sample.

28. FACTORS AFFECT THE RATES OF ENZYME-CATALYZED REACTIONS
Maximal velocity: The rate or velocity of a reaction (v) is the number of
substrate molecules converted to product per unit time. The rate of an
enzyme-catalyzed reaction increases with substrate concentration until a
maximal velocity (V max) is reached . The leveling off of the reaction rate (C
point) at high substrate concentrations reflects the saturation with substrate of
all available binding sites on the enzyme molecules present.

30. Michaelis-Menton Kinetics
• The Michaelis-Menten equation illustrates in mathematical
terms the relationship between initial reaction velocity vi and
substrate concentration [S] . In this model, the enzyme reversibly
combines with its substrate to form an ES complex that
subsequently breaks down to product, regenerating the free
enzyme. The model, involving one substrate molecule, is
represented below:
• where S is the substrate , E is the enzyme ,ES is the enzyme-
substrate complex, P is the product and K1,K-1, K2 are rate
constants.

31. • The Michaelis-Menten equation describes
how reaction velocity varies with substrate
concentration:

32. Important conclusions about Michaelis-Menten kinetics:
• 1. Characteristics of Michaelis Km: Km— characteristic of an
enzyme and its particular substrate, and reflects the affinity of
the enzyme for that substrate.
• Km is equal to the substrate concentration that produce half
Vmax.
• ** Small Km reflects a high affinity of the enzyme for substrate,
because a low concentration of substrate is needed to half-
saturate the enzyme (1/2 Vmax) e.g. Hexokinase
• ** Large Km reflects a low affinity of enzyme for substrate
because a high concentration of substrate is needed to half-
saturate the enzyme. e.g. Glucokinase.

34. • 2. Relationship of velocity to enzyme
concentration: The rate of the reaction
is directly proportional to the enzyme
concentration at all substrate
concentrations.
• 3. Order of reaction: When [S] is much
less than Km the velocity of the
reaction is approximately proportional
to the substrate concentration. The rate
of reaction is then said to be first order
with respect to substrate. When [S] is
much greater than Km the velocity is
constant and equal to Vmax. The rate of
reaction is then independent of
substrate concentration, and is said to
be zero order without respect to
substrate concentration.

35. Lineweaver-Burke plot
• Lineweaver-Burke plot:
• When Vi is plotted against [S], it is not always possible to determine
when Vmax has been achieved, because of the gradual upward
slope of the hyperbolic curve at high substrate concentrations.
• However, if 1/Vi is plotted versus 1/[S], a straight line is obtained
This plot, the Lineweaver-Burke plot (also called a double-reciprocal
plot) can be used to calculate Km and Vmax as well as to determine
the mechanism of action of enzyme inhibitors.

37. • The rate of the reaction is directly proportional to
the enzyme concentration at all substrate
concentrations.

38. • Increase of velocity with
temperature: The reaction
velocity increases with
temperature until a peak velocity
is reached .
• This increase is the result of the
increased number of molecules
having sufficient energy to pass
over the energy barrier and form
the products of the reaction.
• Further elevation of the
temperature results in a decrease
in reaction velocity as a result of
temperature-induced
denaturation of the enzyme.

39. • **Effect of pH on the ionization of the active
site: The concentration of H+ affects reaction
velocity in several ways. First, the catalytic
process usually requires that the enzyme and
substrate have specific chemical groups in
either an ionized or unionized state in order
to interact. For example, catalytic activity
may require that an amino group (-NH3+) of
the enzyme be in the protonated form At
alkaline pH this group is deprotonated, and
the rate of the reaction, therefore, declines.
• ** Extremes of pH can also lead to
denaturation of the enzyme.
• **Also, The pH optimum varies for different
enzymes.

40. Enzyme inhibitors
• Definition: Any substance that can diminish the velocity of an
enzyme-catalyzed reaction is called an inhibitor.
• Reversible inhibitors bind to enzymes through noncovalent bonds.
Dilution of the enzyme-inhibitor complex results in dissociation of
the reversibly bound inhibitor, and recovery of enzyme activity.
• Irreversible inhibition occurs when an inhibited enzyme does not
regain activity on dilution of the enzyme-inhibitor complex.
• The two most commonly encountered types of reversible inhibitors
are competitive and noncompetitive.

41. Competitive inhibitor
• Definition: There is structural similarity between substrate and inhibitor. Both substrate
and inhibitor compete for active site of the enzyme.
• Both substrate (S) and inhibitor (l) can bind with the catalytic site of the enzyme to form
either Enz-S-complex or Enz-l-complex.
• Statin drugs as examples of competitive inhibitors: This group of antihyperlipidemic
agents competitively inhibits the first committed step in cholesterol synthesis. This
reaction is catalyzed by hydroxymethylglutaryl CoA reductase (HMG CoA reductase).
Statin drugs, such as atorvastatin (Lipitor) is structural analogs of the natural substrate
for this enzyme.
• Also, angiotensin-converting enzyme (ACE) inhibitors. They lower blood pressure by
blocking the enzyme that cleaves angiotensin to form the potent vasoconstrictor,
angiotensin II. These drugs, which include captopril, enalapril, and lisinopril, cause
vasodilation and a resultant reduction in blood pressure.

42. Noncompetitive inhibitors
• Definition: This type of inhibition occurs when the inhibitor and substrate
bind to different sites on the enzyme‘
• a) The inhibitor does not alter the catalytic site.
• b) There is no structural similarity between substrate and inhibitor.
• c) The inhibitor can bind either free enzyme or the enzyme-substrate
complex.
• Both enzyme inhibitor complex and enzyme substrate inhibitor complex
are inactive.
• Lead is example of noncompetitive inhibitors that forms covalent bonds
with the sulfhydryl side chains of cysteine in proteins. The binding of the
heavy metal shows non­
competitive inhibition. Ferrochelatase, an enzyme
that catalyzes the insertion of Fe into protoporphyrin (a precursor of
heme), is an example of an enzyme sensitive to inhibition by lead.

46. Regulation of enzyme activity
• Classification of enzymes based on regulation
• 1. Structural enzymes, and housekeeping enzymes: operate close to
equilibrium and are characterized by long half-lives.
• 2. Key or rate-limiting enzymes: These are often allosteric enzymes and are
subject to regulation (activators, inhibitors etc). Many have short half-lives.
These are the key enzymes whose activities control the metabolic pathways.
• There are many ways to regulate enzyme activity
at different levels:

48. 1) Regulation of rate of synthesis or degradation
• Regulation of gene expression controls the quantity and rate of enzyme
synthesis.
• Inducers are certain substances that stimulate synthesis of enzyme by
increasing the rate of gene expression
• Repressors are certain substances that inhibit synthesis of enzyme by inhibition
and decreasing the rate of gene expression of that enzyme.
• Regulation by means of induction/repression of enzyme synthesis is usually
slow in the human, occurring over hours to days.
• Inducers may be the substrate for the enzyme or a compound similar to the
structure of the substrate (gratuitous inducer) or a hormone.
• Repressor may be a product of a chemical pathway or a hormone.

49. • Enzymes subject to regulation of synthesis are often those that
are needed at only one stage of development or under selected
physiologic conditions.
• For example, elevated levels of insulin as a result of high blood
glucose levels cause an increase in the synthesis of key enzymes
involved in glucose metabolism.
• In contrast, enzymes that are in constant use are usually not
regulated by altering the rate of enzyme synthesis.
• Adaptation: The amount of the enzyme inside the cell can be
increased or decreased according to the body needs. For
example , An increase of sugar in the diet will increase the liver
enzymes involved in the carbohydrates metabolism .

51. Allosteric regulation
• Allosteric means other site..
• Allosteric enzymes are regulated by molecules called effectors (also modifiers) that bind
noncovalently at a site other than the active site.
• These enzymes are composed of multiple subunits, and the regulatory site that binds the effector
may be located on a subunit that is not itself catalytic.
• The presence of an allosteric effector can alter the affinity of the enzyme for its substrate, or
modify the maximal catalytic activity of the enzyme, or both.
• Effectors that inhibit enzyme activity are termed negative effectors,
• whereas those that increase enzyme activity are called positive effectors.
• Allosteric enzymes usually contain multiple subunits, and frequently catalyze the committed step
early in a pathway.

53. • Two types of allosteric effectors
• Homotropic effector
• Heterotropic effector
• When the substrate itself serves as an effector, the effect is said to
be homotropic.
• The effector may be different from the substrate, in that case the
effect is said to be heterotropic.
• Heterotropic effectors ,for example, the glycolytic enzyme
phosphofructokinase is allosterically inhibited by citrate, which is
not a substrate for the enzyme .

54. • REGULATION OCCURS AT THE RATE-LIMITING STEP
• Pathways are principally regulated at one key
enzyme, the regulatory enzyme, which catalyzes
the rate-limiting step in the pathway.
• This is the slowest step and is usually not readily
reversible. Thus, changes in the rate-limiting step
can influence flux through the rest of the pathway.

55. FEEDBACK REGULATION and FEEDBACK INHIBITION
• We must, distinguish between feedback regulation, and feedback
inhibition.
• For example, while dietary cholesterol decreases hepatic synthesis of
cholesterol, this feedback regulation does not involve feedback
inhibition.
• HMG-CoA reductase, the rate-limiting enzyme of cholesterol synthesis,
is affected, but cholesterol does not feedback-inhibit its activity.
• Regulation in response to dietary cholesterol or a cholesterol
metabolite inhibits the expression of the gene that encodes HMG-CoA
reductase (enzyme repression).

57. Regulation of enzymes by covalent modification
• In mammalian cells, the two most common forms of covalent modification are partial
proteolysis and phosphorylation. proteolysis constitutes an irreversible modification.
By contrast, phosphorylation is a reversible modification process.
• Many enzymes may be regulated by covalent modification, most frequently by the
addition or removal of phosphate groups from specific serine, threonine, or tyrosine
residues of the enzyme.
• Phosphorylation and dephosphorylation: Phosphorylation reactions are catalyzed by
a family of enzymes called protein kinasesthat use adenosine triphosphate (ATP) as a
phosphate donor. Phosphate groups are cleaved from phosphorylated enzymes by
the action of phosphoprotein phosphatases

58. -- The phosphorylation of the enzyme may activate or inactivate the enzyme
For example, glycogen synthase and glycogen phosphorylase .

59. PROTEASES MAY BE SECRETED AS CATALYTICALLY INACTIVE PROENZYMES
• Certain proteins are synthesized and secreted as inactive precursor
proteins known as proproteins.
• The proproteins of enzymes are termed proenzymes or zymogens.
• Selective proteolysis converts a proprotein by one or more
successive proteolytic “clips” to a form that exhibits the
characteristic activity of the mature protein, eg, its enzymatic
activity.
• Proteins synthesized as proproteins include the hormone insulin
(proprotein = proinsulin), the digestive enzymes pepsin, trypsin,
and chymotrypsin (proproteins = pepsinogen, trypsinogen, and
chymotrypsinogen, respectively), several factors of the blood
clotting and blood clot dissolution cascades , and the connective
tissue protein collagen (proprotein = procollagen).

61. TISSUE ISOZYMES
• Isozymes are fractions of one enzyme having the same catalytic activity
but differ in chemical and immunological structure.
•
• The human body is composed of a number of different cell types that
perform specific functions unique to that cell type and synthesize only
the proteins consistent with their functions.
• Because regulation matches function, regulatory enzymes of pathways
usually exist as tissue-specific isozymes with somewhat different
regulatory properties unique to their function in different cell types.
• For example, hexokinase and glucokinase are tissue-specific isozymes
with different kinetic properties. Also LDH has 5 isoenzymes.

63. ENZYMES in CLINICAL DIAGNOSIS
• Plasma enzymes can be classified into two major groups.
• First, a relatively small group of enzymes are actively secreted into the
blood by certain cell types. For example, the liver secretes zymogens
(inactive precursors) of the enzymes involved in blood coagulation.
• Second, a large number of enzyme species are released from cells
during normal cell turnover. These enzymes almost always function
interacellularly and have no physiologic use in the plasma but the
presence of elevated enzyme activity in the plasma may indicate tissue
damage that is accompanied by increased release of intracellular
enzymes.
• Important examples
• GPT , GOT, CPK, LDH, Amylase, ALP,

64. • Diagnosis of myocardial infarction: Myocardial muscle is the only
tissue that contains more than five percent of the total CK activity as
the CK2 (MB) isoenzyme.
• Appearance of this isoenzyme in plasma is virtually specific for
infarction of the myocardium.
• Following an acute myocardial infarction, this isoenzyme appears
approximately four to eight hours following onset of chest pain, and
reaches a peak of activity at approximately 24 hours .
• [Note: Lactate dehydrogenase activity is also elevated in plasma
following an infarction, peaking 36 to 40 hours after the onset of
symptoms. LDH activity is, thus, of diagnostic value in patients
admitted more than 48 hours after the infarction

66. Thanks