ENZYMES
- Enzymes are biocatalysts. They are all proteins.
Function: Catalzye biological reactions by increasing their rates. It does that by
lowering the free energy of activation.
Free
T* (transition state)
Energy
T*
(G)
with
enzyme
A
(reactant)
B (product)
Progress of reaction
Nomenclature (Naming):
Recommended names: enzymes have the suffix “-ase” attached to the substrate.
Examples: sucrase, urase, glucosidase, etc. or description of type of reaction
performed e.g. lactate dehydrogenase, adenylate cyclase, monoaminooxidase (MAO).
Trivial names:
No relationship to reaction, e.g trypsin pepsin. etc.
Systematic names: complete description of chemical reaction catalyzed.
I.U.B.M.B.: Six major classes:
1. Oxidoreductases: oxidation – reduction reaction.
eg: Lactate + NAD+
LDH
pyruvate + NADH + H+
2. Transferases: transfer of C, N, or P-containing groups. Example:
Serine (CH2) + THF
Glycine + THF
Ser – OH – CH3 - transferase
CH2
3. Hydrolases: cleavage of bonds by addition of water. Example:
Urea + H2O
CO2 + 2NH3
Urease
4. Lyases: cleavage of C-C, C-S and certain C-N bonds (not peptide bonds).
Example:
pyruvate
pyruvate
acetaldehyde
CH3 – C – COOCH3 – CH + CO2
decarboxylase
O
O
5. Ligases: formation of bonds between C and O, S, N. It requires energy (ATP).
Example:
pyruvate
pyruvate
_(CH3) + CO2
carboxylase
ATP
HOOC – CH3 – C – COOO Oxaloacetate
ADP + P
6. Isomerase: racemization of optical isomers. Example:
Methylmalonyl CoA
(MMCoA)
MMCoA mutase
Succinyl CoA
Properties of Enzymes:
(1.) Denaturation: Because enzymes are proteins: they can be denatured by extreme
pH, heat, high concentration of organic solvents, heavy metal ions.
(2). Specificity: high degree of specificity independent of a reaction. c.f. with
inorganic catalyst: no specificity. enzymes catalyze one type of chemical reaction or a
group of closely related reactions. Specificity is due to the presence of:
(3). Active site:
-
Specific pocket within the enzyme.
-
3-dimensional pocket is created by the R-groups of certain a.as residues; that can
only fit the substrate.
-
Induced fit concept:
Binding of the correct substrate induces conformational changes in the enzyme’s
tertiary structure, which bring the catalytic groups in the best alignment for
forming transition state (ES).
Example of. Specificity: chmyotrypsin hydrolyzes peptide bonds of the Phe but
not gly.
(4). Stereospecificity: Example: β-galactosidase attacks derivative of D-galactose.
Trypsin and chymotrypsin: L – a.a. residues.
(5). Catalytic efficiency: 103 – 108 X faster than uncatalyzed reactions.
1 enzyme molecule transforms 100 – 1000 S
P/sec.
-The number of substrate molecules that transformed to product per 1 enzyme
molecule per second is called: turnover number.
(6). Cofactors: non-protein part of the enzyme need for activity.
- Metal ions: Zn2+, Fe2+
- Organic molecules (coenzyme): not tightly bound to enzyme, usually vitamin
derivatives (e.g. NAD, FAD, ).
- Prosthetic group: tightly bound coenzyme, e.g. biotin of carboxylses.
Haloenzyme: protein + cofactor (active).
Apoenzyme: protein (no cofactor) (not active).
(7). Regulation: activation or inhibition according to the need of the cell.
(8). Location: Specific organelles/compartments.
Factors Affecting Reaction Velocity (Rate):
A. Substrate Concentration:
The rate or velocity of a reaction (v): is the number of substrate (s) molecules that is
converted to product (p) per minute (μ moles of product formed perminute).
-
The rate increases with substrate concentration until maximum velocity (Vmax) is
reached, i.e. saturation of binding sites with “S”.
Vmax
Enzymes with single P.P.C.
Allosteric enzymes (multimeric)
[S]
Enzyme Concentration:
At fixed temperature and pH, the rate of enzyme reaction is directly proportional to
enzyme concentration  E, providing E  S.
C. Temperature: (T)
-
V increases with “T” until peak V is reached. The rate of a reaction is doubled by
increase of 10 C in temperature until a maximal velocity is reached. The increase
in temperature increases the number of molecules having sufficient energy to pass
the energy barrier to form product. Further elevation of temperature leads to a
decrease in reaction velocity (V), due to temperature - induced denaturation.
20
40
60
80
Temperature (Cº)
D: pH or H+ : Two effects:
1. Effect on ionic state of the active sitE. Some of the substrates recquire H+.
Therefore, an increase in pH leads to loss of H+ and hence a decline in activity.
2. Effect on enzyme denaturation. Extreme pH alters the ionic state of the R-groups
which play a role in 3-D structure of the enzyme.
-
Most enzymes function optimally at pH near neutrality (exception: pepsin pH 2.5,
arginase 9.7).
Enzyme Kinetics
Michaelis-Menten Equation:
k+1
E+S
ES
k2
E+P
k-1
Vo = Vmax. [S]
Km + [S]
Km is the Michaelis constant = (K – 1 + K2)
K+1
Assumptions:
- [S] >> [E]
- [ES] does not change with time, i.e. the rate of ES formation equals to its rate of
degradation to E + P.
- Initial velocity reaction is used (P formed is very small).
Comments:
1. Characteristics of Km:
- Km2 is characteristic for an enzyme and reflects the affinity of enzyme towards the
substrate.
-
Km numerically = 1 Vmax (does not vary with enzyme concentration).
-
Small Km means high enzyme affinity to substrate. I.e. low [S] is needed to reach
1 V max.
2
- Large Km means low enzyme affinity towards substrate.
2. Velocity (rate) is directly proportional to enzyme concentration.
3. Order of the reaction:
Vmax
[S]>> Km: zero order reaction
Constant
independent of [S]
1
2
Vmax
Km
[S]
- At low [S] << Km : the reaction is a first order (Vα[S]). After that, is a psuedofirst order.
Linewaver – Burke Plot
The previous plot (hyberbolic) is difficult to determine when Vmax is achieved.
Therefore, 1 versus 1 plot gives straight line ( Lineweaver – Burk plot). It is used to
Vo
[S]
Vo
calculate Km and Vmax and determine the mechanism of enzyme inhibition.
1 = Km
+ 1
Vo Vmax [S]Vmax
1
Vmax
-1
Km
1
[S]
Inhibition of Enzyme Activity
The rate of an enzyme-catalyzed reaction can be slowed down by an inhibitor.
Therefore, inhibitors are agents that reduce the rates of enzyme-catalyzed reactions.
Inhibition studies illustrate:
(A) Mode of action of enzymes.
(B) How metabolic reactions are regulated in vivo.
N.B. Denaturation agents are not considered as inhibitors.
Types of Enzyme Inhibition:
-Some inhibitors show some degree of specificity at low inhibitor concentration. Suc
inhibition is:
A. Reversible: enzyme activity is restored by dialysis or chromatogrpahy
B. Irreversible: enzyme activity can not be restored.
-
Reversible inhibition: the inhibitor may be bind non-covalently with the free
enzyme (E) or ES complex or with both. Example: competitive inhibition.
-
Irreversible inhibition: the inhibitor binds covalently with the functional group of
the enzyme. Therefore, the substrate (S) can no longer bind to the enzyme.
Example: esterase inhibitors/ alkylating agents of SH – enzymes.
E - H+ ICH2COOH
E - S- CH2COOH + HI
Metabolic inhibitors are also examples of irreversible inhibitors:
CN - Fe2+, Cu2+, Mg2+
cytochrome oxidase (respiratory chain enzyme) leads to death.
Common Types of Inhibition:
A. Competitive inhibition:
“I” binds reversibly to the active site of “E” and competes with “S”. “I” usually have
structural similarity to “S”. The inhibition is overcome by adding more “S”. i.e. high
“S” concentration.
1.Effect on Vmax: at high [S], Vmax is reached.
2.Effect on Km-: Km is increased. Therefore, more substrate (S) is needed to achieve
1 Vmax.
2
Vmax
1
Vo
+(I)
+ (I)
½ Vmax
Vmax
Km
Km (I)
[S]
-1
Km
-1
Km (I)
1
[S]
Example of competitive inhibitor: oxaloacetate, which competitively inhibits
succinate dehydrogenase.
succinate
SDH
fumarate
malate
CH2-COO-
oxaloacetate
O=C-COO-
CH2-COO-
CH2
COO-
Therefore, oxaloacetate plays a regulatory effector in the TCA cycle, and it binds at
the active site of SDH. Malonate, also acts as a competitive inhibitor of SDH.
B. Non-competitive inhibition
No structural resemblance between “I” and “S”. Binding of “I” at a second specific
site, causes alloesteric conformation change of enzyme. Hence, “S” no longer binds to
the active site. “I” can bind to to “E” or “ES”.
1. Effect on Vmax: Non- competitive inhibition cannot be overcome by increasing
“S” concentration.
2. Effect on Km: “I” does not interfere with “S” binding to “E.” Therefore, Km does
not change. Example: Lead poisoning: Pb – SH – E
Covalent
E is ferrochelatase and δ -ALA dehydrase.
Vmax
1
Vo
Vmax
(I)
+ (I)
½Vmax
1
Vmax (I)
1
Vmax
Km, Km (I)
-1 , -1
Km Km (I)
1
[S]
Regulation of Enzyme Activity:
The substrate concentration inside the cell is almost equal to Km. Therefore; any
increase in [S] prompts an increase in a reaction rate. This leads to return of [S] to
normal.
Mechanisms for Regulating Enzyme Activity:
1.Allosteric binding sites:
- Allosteric (other sites) enzymes are regulated by effectors (modifiers/modulators),
that bind non-covalently at sites other than the active site.
effect on “E” affinity: increase (+ effectors)
: decrease (- effectors)
Notes:
Allosteric enzyme contains multisubunits (multimeric) and usually catalzye the
committed step in a metabolic pathway
- Homotropic effectors: that is when the substrate “S” acts as an effector, usually, it is
a positive effector: i.e. binding of “S” at oen site enhances binding of “S” on the other
site of enzyme (coopratively). Therefore, the enzyme activity curve is sigmoidal.
-
Heterotropic effectors: the effector is different from “S”.
Θ
A
B
C
D
E
Example: Heme formation. Heme acts as a negative effector on δ - ALA synthase.
2. Covalent Modification:
That is addition or removal of phosphate groups from specific ser, thr, or tyr of the
enzyme. Phosphorylation may incresae or decrease enzyme activity.
Example: phosphorylation of glycogen phosphorylation increases its activity, whereas
phosphorylation of glycogen synthetase decreases its activity. (fasting state, fright
reaction).
3. Induction and Repression of Enzyme synthesis:
The amount of cellular enzyme can be regulated leading to an increase (induction) or
decrease (repression) in its activity. Enzymes subject to this type of regulation are
those who often needed during stages of development or under specific physiological
conditions.
For example: increase of plasma insulin levels due to increase in blood glucose
concentration causes an increase in enzymes synthesis of the glycolytic pathway.
(1) and (2) their effects are immediate to minutes while (3) hours to days.
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