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Chapter 3 Enzymes

Almost all processes in the living cell are catalyzed by the specific biocatalyst. Enzymes are catalysts that change the rate of a reaction without being changed themselves. Enzymes are highly specific and their activity can be regulated..

Biocatalyst: enzymes and ribozyme. One of the most important functions of proteins is their role as catalysts. Until recently, all enzymes were considered to be proteins. Several examples of catalytic RNA molecules have now been vertified. Living processes consist almost entirely of biochemical reactions. Without catalysts these reactions would not occur fast enough to sustain life.

Enzymes bind to one or more ligands, called substratee, and convert them into one or more chemically modified products.

Composition of enzymes

Simple enzyme and conjugated enzyme. Conjugated enzyme: apoenzyme + cofactor holoenzyme. Cofactor : prosthetic group+ coenzyme prosthetic group: tightly bond with apoenzyme. FAD, metal, etc. coenzyme loosely bond with apoenzyme. NAD, NADP, etc.

Active site: Each type of enzyme molecule contains a unique, intricately shaped binding surface called an active site. Catalytic residues are highly conserved. Certain amino acids, notably cysteine and hydroxylic, acidic, or basic amino acids, perform key roles in catalysis. Essential group in active site: binding group +catalytic group. Cofactors always be a part of the active site.

Active site
The active site is the region of the enzyme that
binds the substrate, to form an enzyme-substrate complex, and transforms it into product. The active site is a three-dimensional entity, often a cleft or crevice on the surface of the protein, in which the substrate is bound by multiple weak interactions. Two models have been proposed to explain how an enzyme binds its substrate: the lock-and key model and the induced-fit model.

Characteristics and mechanisms


of enzymatic reactions

Characteristics Enzymes have several remarkable properties. First, the rates of enzymatically catalyzed reactions are often phenomenally high. (Rate increases by factors of 106or greater are common.) . Second, in marked contrast to inorganic catalysts, the enzymes are highly specific to the reactions they catalyze. Side products are rarely formed. Finally, because of their complex structures, enzymes can be regulated. This is an especially important consideration in living organisms, which must conserve energy and raw materials.

Specificity: Absolute specificity, relative specificity, and stereospecificity. Activation energy: To proceed at a viable rate, most chemical reactions require an initial input of energy. In the laboratory this energy is usually supplied as heat. At temperatures above absolute zero (-273.1C), all molecules possess vibrational energy, which increases as molecules are heated. Consider the following reaction: A+B C As the temperature rises, vibrating molecules (A and B) are more likely to collide, A chemical reaction occurs when the colliding molecules possess a minimum amount of energy called the activation energy.

Activation energy
Uncatalyzed activation energy Non-enzymatic activation energy

Energy

Enzymatic activation energy

Substrate

Total energy

Changes of reaction

Product

Progress of reaction

Not all collisions result in chemical reactions because only a fraction of the molecules have sufficient energy. Induced-fit hypothesis and transition state. Substrates induce conformational changes in enzymes. During any chemical reaction reactants with sufficient energy will attain transition state (a strained intermediate form) when the substrate binds to the enzyme (inducing).

Induced-fit Theory

substrate

Complex of substrate-enzyme enzyme

Mechanisms Proximity effect and orientation arrange: For a biochemical reaction to occur, the substrate must come into close proximity to catalytic functional groups (side chain groups involved in a catalytic mechanism ) within the active site. In addition, the substrate must be precisely, spatially oriented to the catalytic groups. Once the substrate is correctly positioned, a change in the enzymes conformation may result in a strained enzymesubstrate complex. This strain helps to bring the enzyme-substrate complex into the transition state.

Multielement catalysis (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. These groups are referred to as general acids or general bases. Surface effect: The strength of electrostatic interactions is related to the capacity of surrounding solvent molecules to reduce the attractive forces between chemical groups. Water is largely excluded from the active site as the substrate binds.

Enzyme kinetics

The rate or velocity of a biochemical reaction is defined as the change in the concentration of a reactant or product per unit time. Plotting initial velocity v versus substrate concentration [S].The rate of the reaction is directly proportional (first order reaction) to substrate concentration only when [S] is low. When [S] becomes sufficiently high that the enzyme is saturated, the rate of the reaction is zero-order with respect to substrate.

Michaelis-Menten Equation
k1 S +E k2 ES k3 E+P
(1)

K1= rate constant for ES formation K2= rate constant for ES dissociation K3= rate constant for product formation and release from the active site

v=

Vmax [S] Km + [S]


(2)

k1 S +E k2

ES

k3

E+P
(3)

ES formation = K1 ( [E] - [ES] ) [S] ES dissociation = K2 [ES ]+ K3 [ES]

(4)

K1 ( [E] - [ES] ) [S] = K2 [ES ]+ K3 [ES] ( [E] - [ES] ) [S]


=

K2+ K3

[ES]

K1

Michaelis and Menton introduced a new constant, Km ( now referred as the Michaelis constant): K2+ K3 Km=

K1
( [E] - [ES] ) [S]

Km =
[ ES]

Km [ES] = [E] [S] [ES] [S]


Km [ES] + [ES] [S] = [E] [S] [ES] ( Km + [S] ) = [E] [S] [E] [S]

[ES] =
Km+[S]

(5)

Since V= K3 [ES], from ( 5 )

[E] [S] V= K3 (6)

Km+[S]
When the [S] is much higher than the enzymes, all enzymes form [ES], that is, [E]= [ES], and maximum velocity ( Vmax ) can attain.

Vmax = K3 [ES] = K3 [E] Vmax K3 = [E]

(7)

Vmax [E] [S] V= [E] Km+[S] =

Vmax [S] (2) Km+[S]

Significances of Km and Vmax


1) When [S] = Km, Vmax [S] Vmax V= = [S] + [S] 2 2) When [S] is very much greater than Km, Vmax [S] Vmax [S] V= = = Vmax Km+[S] [S]

3) It may reflect the affinity of the enzyme for its


substrate. If K3 is much smaller than K2, that is K3 K2, Km is the dissociation constant for the [ES]. K2 K m= K1 4) From Vmax = K3 [ES] = K3 [E], enzymes are saturated. Vmax K3= [E] The turnover number (Kcat ) = K3. This quantity is the number of moles of substrate converted to product each second per mole of enzyme.

Lineweaver-Burk Double-reciprocal plot


Km 1 1 1 + v = V Vmax max [S]

y = mx + b

Km 1 1 1 + v = V Vmax max [S]

Slope

(intercept on the vertical axis)

(intercept on the horizontal axis)

Multiple factors affect the rates of enzyme-catalyzed reactions.


Temperature
While raising temperature increases the rate of an enzyme-catalyzed reaction, this holds only over a strictly limited range of temperatures. The reaction rate initially increases as temperature rises owing to increased kinetic energy of the reacting molecules. Eventually, however, the kinetic energy of the enzyme exceeds the energy barrier for breaking the weak bonds that maintain its secondary-tertiary structure. At this temperature, denaturation, with an accompanying precipitate loss of catalytic activity, predominates.

Enzymes from humans, who maintain a body temperature of 37 C, generally exhibit stability at temperature up to 45-55 C. Enzymes from microorganisms that inhabit natural hot springs or hyperthermal vents on the ocean floor may be stable at or above 100 C. Optimum temperature: Temperature at which it operates at maximal efficiency.

Enzyme activity

Temperature ( C )

pH When enzyme activity is measured at several pH values, optimal activity typically is observed between pH values of 5 and 9. However, a few enzymes are active at pH values well outside this range. pH optimum: The pH value at which an enzymes activity is maximal is called the pH optimum.

Initial rate is proportionate to enzyme concentration The initial rate of a reaction is the rate measured before sufficient product has been formed to permit the reverse reaction to occur. The initial rate of an enzyme-catalyzed reaction is always proportionate to the concentration of enzyme. Note, however, that this is statement holds only for initial rates. Substrate concentration


[S]>>[E] [E]v

pH dependent of enzyme activities

Enzyme activity

Pepsin

Amylase

Acetylcholinesterase

pH

(4)

Enzyme inhibition

The activity of enzymes can be inhibited. Many substances can reduce or eliminate the catalytic activity of specific enzymes. Inhibition may be irreversible or reversible. Irreversible inhibitors usually bond covalently to the enzyme, often to a side chain group in the active site. For example, enzymes containing free sulfhydryl groups can react with alkylating agents such as iodoacetate and heavy metals. This process is not readily reversed either by removing the remainder of the free inhibitor or by increasing substrate concentration. Specific inhibitor: specifically bind to essential amino acid on active site. Some organic phosphor compounds could specifically bind to OH of serine.

Non specific inhibitor: not only binds to essential group, but also to outsides of essential group. Hg2+, Ag2+ and As3+ . In reversible inhibition: the inhibitor can dissociate from the enzyme because it binds through noncovalent bonds. The most common forms of reversible inhibition are competitive and noncompetitive.

1)

Competitive inhibition

Competitive inhibitors typically resemble the substrate Classic competitive inhibition occurs at the substrate-binding (catalytic) site. The chemical structure of a substrate analog inhibitor (I) generally resembles that of the substrate (S). It therefore combines reversibly with the enzyme, forming an enzyme-inhibitor (EnzI) complex rather than an EnzS complex.

Competitive inhibition

E+S +

ES

E+P

I
Ki EI v=

Vmax [S]

[I] )) + [S] Km (1+


Ki

Km [I] ) ) 1 1 1 (1+ + v = V Ki [S] Vmax max

inhibitor
No inhibitor

Noncompetitive inhibition
In noncompetitive inhibition, no competition occurs between S and I. The inhibitor usually bears little or no structural resemblance to S and may be assumed to bind to the enzyme at a site other than the active site. Both EI and EIS complexes form. Inhibitor binding alters the enzymes three-dimensional configuration and blocks the reaction.

Noncompetitive inhibition

E+S + I Ki EI + S

ES + I Ki ESI

E+P

Plots of 1/V versus 1/[S] in the presence of several concentrations of the inhibitor intersect at the same point on the horizontal axis, -1/Km. In noncompetitive inhibition the dissociation constants for ES and EIS are assumed to stay the same.

inhibitor

No inhibitor

3)

Uncompetitive inhibition

The inhibitor bind to ES and results in decrease of both ES and P (also free E). E+S ES E+S + I Ki ESI

Uncompetitive inhibition

E+S

ES + I Ki ESI

E+P

1 Km 1 1 I (1 ) v Vm ax S Vm ax Ki

inhibitor No inhibitor

4) Effect of activator on the enzyme activities


Activator: substances enable non-active enzyme to become active one. Metals such as Mg2+, K+, Mn2+, etc. Essential activator and non-essential activator.

5) Enzyme activity assay and unit of enzyme activity


Enzyme activity is measured in international units (I.U.) One I.U. is defined as the amount of enzyme that produces 1mol of product per minute. An enzyme specific activity, a quantity that is used to monitor enzyme purification, is defined as the number of international units per milligram of protein. A new unit for measuring enzyme activity called the katal, has recently been introduced. One katal (kat) indicates the amount of enzyme for the transformation of 1 mole of substrate per second. 1 IU =16.6710-9 kat

4 Regulation of enzyme
The thousands of enzyme-catalyzed chemical reactions in living cells are organized into a series of biochemical or metabolic pathways. Each pathway consists of a sequence of catalytic steps. The product of the first reaction becomes the substrate of the next and so on. Metabolic and other processes are controlled by altering the quantity or the catalytic efficiency of enzymes.

1) Regulation of enzyme activities


A. Proenyme or Zymogen: Certain proteins are manufactured and secred in the form of inactive precursor proteins known as proproteins. When the proteins are enzymes, the proproteins are termed proenzymes or zymogens. Conversion of a proprotein to the mature protein involves selective proteolysis, a process that converts the proprotein by one or more successive proteolytic clips to a form having the characteristic activity of the mature protein ( its enzymatic activity ). Examples include the hormone insulin (proinsulin), pepsinogen, trypsinogen, etc.

Selective proteolysis of a proenzyme may be viewed as a process that triggers essential conformational changes that create the catalytic site.

B. Allosteric enzyme

Allosteric enzymes are enzymes whose activity at the catalytic site may be modulated by the presence of allosteric effectors at an allosteric site. Allosteric effector could be products, substrate, and so on. Feed back inhibition referred to the inhibition of the activity of an enzyme in a biosynthetic pathway by an end product (often as allosteric effectors) of that pathway.

C. Regulatory covalent modification


Regulatory covalent modifications can be reversible or irreversible. In mammalian cells, the two most commonly used forms of covalent modification are partial proteolysis and phosphorylation. Because cells lack the ability to reunite the two portions of a protein produced following hydrolysis of a peptide bond, the partial proteolysis is considered an irreversible modification.

Hydrolysis of the phosphoesters formed when a protein is covalently phosphorylated on the side chain of a serine, threonine, or tyrosine residues is both thermodynamically spontaneous and readily catalyzed by enzymes called protein phosphatases. Hence, phosphorylation represents a reversible modification process.

Cyclic phosphorylation and dephosphorylation is a common cellular mechanism for regulating protein activity. In this example, the target protein R (orange) is inactive when phosphorylated and active when dephosphorylated; the opposite pattern occurs in some proteins.

2) Regulation of enzyme quantity


Rate of synthesis and degradation determine enzyme quantity. The quantity of an enzyme in a cell may be increased either by elevating its rate of synthesis, by decreasing its rate of degradation, or by both. Cells can synthesize specific enzymes in response to changing metabolic needs, a process referred to as enzyme induction. The induction accomplished by genetic control. Although many inducers are substrates for the enzymes they induce, compounds structurally similar to the substrate may be inducers but not substrates. Conversely, a compound may be a substrate but not an inducer.

The synthesis of certain enzymes may also be specifically inhibited. In a process called repression, the end product of a biochemical pathway may inhibit the synthesis of a key enzyme in the pathway. Both induction and repression involve cis-elements, specific DNA sequences located upstream of genes that encode a given enzyme, and a trans-acting regulatory proteins. Regulation of enzyme degradation. The degradation of mammalian proteins by ATP and ubiqitin-dependent pathways and by ATPindependent pathways. It also Related to the nutrition and hormone state.

Compartmentation

In eukaryotic cells, biochemical pathways are segregated into different organelles. One purpose for this physical separation is that opposing processes are easier to control if the occur in different compartments. For example, fatty acid biosynthesis occurs in the cytoplasm, while the energy-generating reactions of fatty acid oxidation occur within the mitochondria. Another factor is that each organelle can concentrate specific substances such as substrates and coenzymes. In addition, special microenvironments are often created within organelles.

3) Isoenzymes
The enzymes catalyzing the same biochemical reaction. Lactate dehydrogenase (LDH)

Isoenzymes

H subunit

M subunit

Isoenzymes of lactate dehydrogenase

5 Nomenclature and classification


The International Union of Biochemistry (IUB) adopted a complex but unambiguous system of enzyme nomenclature based on reaction mechanism. (1) Reactions and the enzymes that catalyzed them form six classes, each having 4-13 subclasses.

(2) The enzyme name has two parts. The first names the substrate or substrates. The second, ending in ase, indicates the type of reaction catalyzed. (3) Additional information, if needed to clarify the reaction, may follow in parentheses; eg, the enzyme catalyzing
L-malate + NAD+ pyruvate + CO2 + NADH + H+

is designated 1.1.1.37 L-malate: NAD+ oxidaoreductase (decarboxylating). (4) Each enzyme has a code number (EC) that characterizes the reaction type as to class, subclass, and subsubclass.

Classification
Six classes based on reaction mechanism: (1) Oxidoreductases: LDH, Cytochrome C, etc. (2) Transferases: methyl transferase. (3) Hydrolases: amylase (4) Lyases removing a group to form a double bond, or reverse reaction. (5) Isomerase to catalyze the intertransfer of isomers. (6) Ligase. catalyzing two substrates link to form one compound.

Relationship between Enzyme and Medicine

1.

A
B C

D
E

2
A

B
C D

3. ( A. B. C. D. E.

4. Holoenzyme refer to (

A. Complex of enzyme with substrate B. Complex of enzyme with suppressant C. Complex of enzyme with cofactor D. Inactive precursor of enzyme E. Complex of enzyme with allosteric effector

5. ( A. B.

C.
D. E.

6. (

A. B.

C.
D. E.

7. ( A.

B.
C. D. E.

8. Michaelis-Menten enzyme kinetics diagram of curves is a ( ) A. straight line B. rectangular hyperbola C. S shape curve D. parabola E. Not above all

9. Km( A. Km

B. 1/Km,

C. Kmmmol/min
D. Km E. Km

10. (

A.
B. C. ,

D. ,
E.

11. In anticompetitive inhibition of enzyme, the reaction kinetics parameter change as ( ) A. Km,Vmax invariably B. Km,Vmax C. Km invariably,Vmax D. Km,Vmax invariably

E. Km,Vmax

12. (
A. B. -

C.
D. E. -

13.

(
A.

B. , C. D. , E. ,

14.

SH is one enzymes essential group. Which substance can protect this enzyme from oxidation?

A. Cys
B. GSH C. urea D. ionic detergent E. ethanol

15. (

A.
B. C.

D.
E.

16. The characteristic constants of enzymes include ( ) A. Enzymic optimum temperature B. Enzymic optimum pH C. Vmax D. Km

E. KS

17. (

A. B. C. D. E.

18. Cofactors of enzyme are (

A. Micromolecule organic compounds B. metal ion C. vitamine D. various kinds of organic and inorganic compounds E. A kind of conjugated protein

19. -SH,( ) A. GSH


B. C C.

D. A
E.

20. (

A.
B. C.

D.
E.

Thank you!

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