 Introduction
 Classification of enzymes




3. Properties :
3.1. Kinetics : Michaelis-Menton &Lineweaver-Burk Plots
3.2. Inhibition : Competitive, Uncompetitive & Non-competitive
3.3. Catalysis : Proximity, electrostatic, acid-base, covalent
 4. Cofactors and coenzymes
 5. Temperature & pH effects on reactions
 6. Application in Industry and medicine


1.
2.

Enzymes are usually proteins of high molecular weight that act as catalysts
How enzymes work?
recognize very specific substrates, perform specific chemical reactions high speeds.

 Enzymes make and break specific chemical bonds of the substrates at a faster rate without being consumed in the process.
 At the end of each catalytic cycle, the enzyme is free to begin again with a new substrate molecule

Active site = a part of an enzyme where substrate bind and undergo a chemical reaction

 Enzymes are generally globular proteins, some are simple proteins and others are conjugated proteins.
 Enzymes catalyze reactions without bring destroyed or chemically changed.
 Enzymes are usually very specific as to which reactions they catalyze and the substrates that are involved in these reactions.
 Example: maltase only catalyze the hydrolysis of maltose and lipase catalyze the hydrolysis of oil.

 Enzymes are usually proteins of high molecular weight that act as catalysts
 Catalysts = substance that enhances the rate of a chemical reaction but is not permanently altered by the reaction
 Catalysts decrease the activation energy required for a chemical reaction (provide an alternative pathway that require less energy)

 Transition state occurs at the apex of both reaction pathway
 Reaction catalyze by enzyme require low activation energy to convert reactant (substrate) from the ground state to the transition state
Transition state
Ground state
(stable, low energy form of mol.)

 Each type of enzyme molecule contain a unique, intricately binding surface called active site
 Substrate bind to active site which is typically small cleft on a large protein mol.
 Beside being binding site, amino acid side chains that line the active site actively participate in catalytic process

 Introduce by Emil Fischer 1890
 illustrate enzyme specificity
 Each enzyme binds to a single type of substrate (bcoz the active site and the substrate have complementary structure)
 active site = substrate binding surface



 Modification of lock and key model by Daniel
Koshland (1958)
 Flexible structure of protein is taken into account
 In this model, substrate does not fit precisely into a rigid active site
 Noncovalent interaction between enzyme & substrate change the active site structure, make it fit for the substrate

 Enzymes named according to the type of chemical reaction it catalyzes
 Six major enzyme categories :
1 )Oxidoreductase
- Catalyze oxidation-reduction (redox) reaction
- Subclasses – dehydrogenases, oxidases, oxygenases, reductases, peroxidases & hydroxylases

2) Transferases
- catalyze reactions that involve the transfer of groups from one molecule to another
- eg. Transaminases, transcarboxylases
3) Hydrolase
- cleave bonds by adding water
- eg. Phosphatases, peptidases, esterase

4) Lyases
- catalyze reactions in which groups (eg. H
2
0,
CO
2
, NH
3
) removed to form a double bond
- eg. Decarboxylases, hydratases, deaminases
5) Isomerases
- catalyze intramolecular rearrangements --- eg. epimerases or mutases

6) Ligases
- catalyze bond formation between two substrate molecules
- eg. Synthetase, carboxylase

 Enzyme kinetics is the quantitative study of enzyme catalysis (chemical reaction catalyzed by enzyme).
 Kinetic studies measure reaction rates and the affinity of enzymes for substrates and inhibitors .
 Studying an enzyme's kinetics in this way can reveal the catalytic mechanism of this enzyme, its role in metabolism , how its activity is controlled, and how a drug or a poison might inhibit the enzyme.






For a given enzyme concentration and for relatively low substrate concentrations, the reaction rate increases linearly with substrate concentration the enzyme molecules are largely free to catalyze the reaction increasing substrate concentration means an increasing rate at which the enzyme and substrate molecules encounter one another.
However, at relatively high substrate concentrations, the reaction rate is zero-order with respect to substrate. the enzyme active sites are almost all occupied

 Model to investigate enzyme rates
 Proposed by Leonor Michaelis and Maud Menten in 1913
 When substrate (S) binds in the active site of enzyme (E), an intermediate complex (ES) is formed
 During transition state, substrate is converted into product
 Later, product dissociates from enzyme

 Rate equations for an enzyme catalyzed reaction support a theory involving the formation of ES complexes.
 At high [S], S saturates E, and the reaction rate is independent of the [S].
 The value of v under this condition is called the maximum velocity, Vmax. At low [S], the reaction is first-order with respect to S. The plot of v versus [S] from low to high [S] is a rectangular hyperbola. The rate equation ( Michaelis-Menten equation ) that describes this relationship is

 The concentration of substrate that corresponds to half-maximum velocity is called the Michaelis constant, Km. The enzyme is half-saturated when [S] = Km.
Michaelis-Menten plot

When [S] << Km the velocity will be given by v = V max [S]/ Km . The velocity depends linearly on [S]. Doubling [S] doubles the rate.
When [S] >> Km,
The equation reduces to v = V max, the velocity approaches V max , and the dependence of velocity on substrate concentration approaches a horizontal line
If [S] = Km , the velocity will be one-half of V max.
When there is no substrate present ([S] = 0), there is no velocity

 Activity of an enzyme can be inhibited
 Molecules that reduce enzyme activity = inhibitors
(drugs, antibiotics, food preservatives, poisons)
 Occur when a compound competes with substrate for the active site of the free enzyme
 3 classes of enzyme inhibitors : competitive inhibitors noncompetitive inhibitors uncompetitive inhibitors

 Competitive inhibitors bind reversibly to free enzyme, not the ES complex, to form an enzyme-inhibitor (El) complex.
Substrate and inhibitor compete for the same site on the enzyme

Competitive inhibitor interfere with active site of enzyme so substrate cannot bind

 Substance that behave as competitive inhibitors, reduce enzyme affinity for substrate
 Enzyme activity decline, no reaction occur when EI complex exists
 Effect of competitive inhibitors on enzyme activity is reversed by increasing [S]
 At high [S], all active site filled with substrate

 The inhibitors binds only to the enzymesubstrate complex (ES), and not the free enzyme

 In some enzyme-catalyzed reactions, an inhibitors can bind to both enzyme and enzyme-substrate complex (EI)

 A noncompetitive inhibitor binds at a site other than the active site of the enzyme
 Inhibitor binding result in a modification of enzyme’s conformation that prevent product formation
 Noncompetitive inhibitors do not affect substrate binding & have little structural resemblance to substrate
 Is only partially reversed by increasing the substrate concentration

 Enzyme catalysis is the catalysis of chemical reactions by specialized proteins known as enzymes
 Enzyme achieve higher catalytic rates because their active site possess structure that uniquely suited to promote catalysis
 Factors contribute to enzyme catalysis :
 proximity and strain effects
 electrostatics effects
 acid-base catalysis
 covalent catalysis

(1) proximity and strain effects
 For a biochemical reaction to occur, the substrate must closely approach the catalytic site with proper orientation



Once substrate correctly positioned, result in a strained enzyme-substrate complex.
This strain help to bring the enzyme-substrate complex into the transition state
When an enzyme and substrate are in very close proximity, they behave as if they are part of the same molecule

(2) electrostatic effects
 the strength of electrostatic interactions is related to the capacity of surrounding solvent molecules to reduce the attractive forces between chemical groups.
 the dielectric constant near the active site is often low, this may influence the chemical reactivity of the substrate.
 weak electrostatic interactions in both the active site and the substrate, are believed to contribute to the catalysis.

(3)Acid-Base catalysis
 Chemical groups can often be made more reactive by adding or removing a proton.
 Enzyme active sites contain side chain groups that act as proton donors or acceptors.

(4) Covalent catalysis
 In some enzymes a nucleophilic side chain group forms an unstable covalent bond with the substrate



The enzyme-substrate complex then forms product
The covalent bond must, at a later stage in the reaction, be broken to regenerate the enzyme.
This mechanism is found in enzymes such as proteases like chymotrypsin and trypsin, where an acyl-enzyme intermediate is formed.

•Examples of covalent bond formation between enzyme and substrate.
•In each case, a nucleophilic center (X:) on an enzyme attacks an electrophilic center on a substrate.

 Catalytic activity of some enzymes depends only on interaction between active site amino acids and substrate
 other enzymes require non protein compounds for their activities (cofactor)
 Apoenzyme = protein component of an enzyme that lacks an essential cofactor
 Holoenzyme = intact enzyme with bond cofactor

 Cofactor = non protein chemical compound that is bound to enzyme
 2 types of cofactor metal cations (Mg 2+ , Zn 2+ ) coenzymes

Metals cations






2 classes transition metals (Fe 2+ , Cu 2+ ) alkali and alkaline earth metals
(Na + , K + , Mg 2+ , Ca 2+ )
Metal ions provide a high concentration of +ve charge, is useful in binding small molecules
Transition metals are most often used in catalysis
Transition metals act as lewis acid (electron pair acceptors)
Help to orient the substrate within the active site.
The substrate-metal ion complex polarizes the substrate and promotes catalysis

Coenzymes
 Most derived from vitamins
 Vitamin =organic nutrients required in human body in small amounts
 2 classes – water soluble & lipid soluble
 Vitamin-like nutrients (eg. Lipoic acid, carnitine) that can be synthesized in small amounts, facilitate enzyme-catalyzed reaction

Vitamins and their coenzymes forms
Vitamin
Water Soluble vitamin
Thiamine (B
1
)
Riboflavin (B
2
)
Pyridoxine
Nicotinic acid
Lipid soluble vitamins
Vitamin A
Vitamin D
Coenzyme form
Thiamine pyrophospophate
FAD and FMN
Pyridoxal phosphate
NAD and NADP
Retinal
1,25-Dihydroxycholecalciferol
Reaction
Decarboxylation
Redox
Amino group transfer
Redox
Vision, growth
Calcium and phosphate metabolism

Effect of temperature and pH on enzymecatalyzed reactions
 Any environmental factor that disturb proteins structure may change enzymatic activity
 Enzymes sensitive to changes in temp and pH

Temperature
 All chemical reactions affected by temperature
 The higher temperature = the higher reaction rate
 Reaction velocity increase as more molecules have sufficient energy to enter transition state
 The rate of enzyme-catalyzed reaction also increase with increasing temp.
 However, enzymes are protein that denatured at high temperature

 Each enzymes has an optimum temperature at which it operate at maximal efficiency


If temperature raised beyond it optimum temperature, enzyme activity will declines
An enzyme’s optimum temp usually close to the normal temp. of an organism it comes from

pH
 Hydrogen ion affects enzymes in several ways
 Changes in [H] ion can affect the ionization of active site groups.
 Eg. Catalytic activity of certain enzymes require protonated form of side chain amino group
 If the pH become sufficiently alkaline, the group will lose it proton, enzyme activity will depressed
 Eg. If a substrate contains an ionizable group, a change in pH may alter its capacity to bind to the active site.
 Changes in ionizable groups may change the tertiary structure of the enzyme.
 Drastic changes in pH often lead to denaturation

 Few enzymes can tolerate large changes in pH, but most enzymes are active only within a narrow pH change
 pH value at which enzyme’s activity is max is called pH optimum