Enzymes

Enzymes Biocatalyst synthesized in the living cells, mostly protein in nature,thermolabile in character and specific in their action.Enzymes are proteins functioning as catalysts that speed up reactions by loweringthe activation energy.The enzyme catalysts regulate the fuction of cells and organisim,
Classification: a Intracellular enzymes b Extra cellular enzymes


Properties of enzymes -Enzymes can act on only one type of substrate (specific) -They always produce the same products -Although they take part in the reaction ,they are not used up -Enzymes are dynamic protein -Because enzymes are proteins they are denatured by heat or some chemicals -Most enzymes are soluble in water -enzymes are colloidal in nature Small quantity is required for enzyme action

According to international union of biochemistry (IUB) divided into six major groups: 1-Oxidoreducatase Oxidation reduction reaction 2-Transferase catalyze the transfer of functional group 3-Hydrolase hydrolysis of various compound 4-Lyases specialized in addition or removal of water ,ammonia, CO2. 5-Isomerase all isomerization reaction. 6- Ligases catalyses the synthetic reaction According to the reaction being catalyzed.
Another classification: that each class in turn is subdivided into many subclass which are further divided. A four digit enzyme commission(E.C.)


Enzymes also classified on the basis of their composition . Enzymes wholly of proteins are known as simple enzyme, in contrast to complex enzyme which are composed of protein+small organic molecule. Complex enzyme ------------holoenzyme. Protein component in complex enzyme apoenzyme. Non protein component is called coenzyme or prosthetic group

Structure of enzymes

Enzymes
Simple (only protein)
Complex or holo_enzymes (protein part and non protein part – cofactor) Non protein part Cofactor
Apoenzyme (protein part)
Coenzyme -large organic molecule -loosely bound to apoenzyme
Prosthetic groups usually small inorganic molecule or atom; usually tightly bound to apoenzyme



Apoprotein – Responsible for the reaction.Cofactors –responcible for :a. Bond formation between enzyme and substrateb. Transfer of functional group.c. Takes place in the formation of tertiary structure of protein part.Cofactors(nonprotein part):1. Prosthetic group(when cofactor very tightly bound to aopenzyme and has small size .2. Metal ion .3. Coenzyme (organic molecule derived from the vitamin B which participate directly in enzymetic reactions)

Prosthetic group. Heme group of cytochrome.. Biotin of acetyl-CoA CARBOXYLASE.Metal ionsFe –cytochrome oxidase, catalaseCu – cytochrome oxidase, catalaseZn – alcohol dehydrogenaseMg – hexokinase, glucose-6-phosphataseK, Mg –pyruvate kinaseNa, k –ATP-ase Coenzyme TPP, FMN, FAD, pantothenic acid, COA, NAD,NADPH Enzymes that require a metal in their composition known as metalloenzymes

Factors affecting enzyme activity: 1.Concentration of enzyme. 2. Concentration of substrate. 3.Effect of Temperature. 4.Effect of PH. 5. Effect of product concentration. 6. Effect of activator.


1. Conc. Of enzyme: as the conc. Of enzyme is increased ,the velocity of the reaction is increased.


2 Conc. Of the substrate: increase in the substrate conc. gradually increase the velocity of enzyme reaction within the limited range of substrate level. distinct phases of reaction are observed in the graph: a/ at a low substrate concentration the velocity of reaction is directly proportional to the substrate level (phase A). b/In the second phase (phase B) it is not directly proportional to the enzyme activity.

c/ In the third phase (phase C ) the reaction is independent of the substrate concentration.

The Michaelis-Menten Equation is the Fundamental Equation of Enzyme Kinetics

Louis Michaelis and Maud Menten's theory. It assumes the formation of an enzyme-substrate complex. It assumes that the ES complex is in rapid equilibrium with free enzyme. Breakdown of ES to form products is assumed to be slower than 1) formation of ES and 2) breakdown of ES to re-form E and S. Briggs and Haldane later introduced the steady state assumtion.

The Michaelis-Menten Equation is the Fundamental Equation of Enzyme Kinetics

E = enzyme concentration. S = Substrate concentration. ES = Enzyme-substrate complex concentration (noncovalent). P = product concentration. k1 = rate constant for formation of ES from E + S. k-1 = rate constant for decomposition of ES to E + S. k2 = rate constant for decomposition of ES to E + P.
E + S ES E + P
k1
k2
k-1

Development of the Michaelis-Menton Equation

1. The overall rate of product formation: v = k2 [ES] 2. Rate of formation of [ES]: vf = k1[E][S] 3. Rate of decomposition of [ES]: vd = k-1[ES] + k2 [ES] 4. The steady state assumption requires that: Rate of ES formation = Rate of ES decomposition 5. So: k1[E][S] = k-1[ES] + k2 [ES]
E + S ES E + P
k1
k2
k-1


6. In solving for [ES], use the enzyme balance to eliminate [E]. ET = [E] + [ES] 7. k1 (ET - [ES])[S] = k-1[ES] + k2 [ES] k1 ET[S] - k1[ES][S] = k-1[ES] + k2 [ES] 8. Rearrange and combine [ES] terms: k1 ET[S] = (k-1 + k2 + k1 [S])[ES] k1 ET[S] 9. Solve for [ES] = ----------------------- (k-1 + k2 + k1 [S])
Michaelis-Menton Derivation


ET[S] 10. Divide through by k1: [ES] = ----------------------- (k-1 + k2)/k1 + [S] 11. Defined Michaelis constant: KM = (k-1 + k2) / k1 12. Substitute KM into the equation in step 10. 13. Then substitute [ES] into v = k2 [ES] from step1 and replace Vmax with k2 ET to give:
Vmax[S] vo = ----------- KM + [S]
Michaelis-Menton Derivation


3.Effect of Temp: velocity of an enzyme reaction increased with increase in Temp. up to maximum and decline. A bell shape curved is usually observed.



The optimum temp. for most of the enzymes is between (40-45)C. When enzymes are exposed to a Temp above 50C denaturation occurs leading to dearrangment in the tertiary structure of protein.

Understanding Km

The "kinetic activator constant" Km is a constant. Km is a constant derived from rate constants. Km is, under true Michaelis-Menten conditions, an estimate of the dissociation constant of E from S.A measure of ES binding.Small Km means tight binding; large Km means weak binding. Where k2 is small then Km ≈ Kd.

4. Effect of PH: increase in (H) ion concentration(PH) influence the enzyme activity and bell- shaped curve is normally obtained


5-Effect of product concentration: the accumulation of reactive product generally decrease the enzyme velocity . For certain enzymes , the product combine with active site of the enzyme and form a loose complex and this inhibit the enzyme activity.
6.Effect of activator: Some enzymes require certain inorganic metallic cation, Mg+2,Mn+2,Zn+2,Ca+2. for their optimum activity, these metals function as activator of the enzyme velocity through various mechanism
Each enzyme has own optimum PH at which the velocity maximum. Most of the enzyme showed optimum activity around neutral PH , PH(6-8)


Two group of enzyme requiring metals for their activity are distinguished; A: metal-activated enzyme: metal is not tightly held by enzyme and can be exchanged easily.EX: ATPase(Mg+2,Ca+2). B: Metalloenzymes: enzymes hold tightly and not exchangable:Ex: alcohol dehydrogenase,Alkaline phosphatase


Enzyme Inhibition: Enzyme Inhibitor: a substrate which binds with enzyme and bring about a decrease in catalytic activity of that enzyme Enzyme Inhibitor 3 groups Reversible Irreversible Allosteric
Inhibitors are chemicals that reduce the rate of enzymic reactions. They are usually specific and they work at low concentrations. They block the enzyme but they do not usually destroy it. Many drugs and poisons are inhibitors of enzymes in the nervous system.
Enzyme Inhibitors


Meaning of enzyme inhibitors Irreversible inhibitors : Inhibition of enzyme activity by combining with active site. Reversible inhibitors : Inhibitors binds non-covalently with the enzyme and the enzyme can be reversed if the inhibitor is removed. a-Competitive inhibitors : Inhibition of enzyme activity by competing with active site . b-Un Competitive inhibitors : Inhibition of enzyme activity by combining with allosteric site. c-Non Competitive inhibitors : Inhibition of enzyme activity by combining with both to free enzyme and ES at allosteric site.

1-Reversible Inhibitor: Inhibitor binds non-covalently with enzyme and the enzyme inhibition can be reversed if the inhibitor is removed. Reversable
Competative inhibitor
non- competative inhibitor
Un-competative inhibition

Competitive I binds at the active site VM does not change KM changes

The inhibitor competes with the substrate and binds at the active site of the enzyme but doesn't undergo any catalysis. Ex. Of clinical &pharmacological inhibition.
xanthine
Uric Acid
Xanthine oxidase
Allopurinol
Hypoxanthine
Allopurinol acts as competitive inhibitor to xanthine oxidase , it competes with enzyme xanthine oxidase and prevent or block the reaction. so control Gout ( decrease uric acid production).
a.Competative inhibition: The inhibitor is closely resemble the substrate {S} substrate analogue



Dihydrofolic acid
Tetrahydrofolic acid FH4 Folinic acid
Dihydrofolate Reductase

methotrxate

Antimetabolites are chemical compound that block the reaction by their inhibitor effect on the enzyme . Antimetabolite are structural analogues of substrate and they are a competitive inhibitor . They are used for cancer therapy


b/Non-competitive Inhibitor: The inhibitor binds at the site other than the active site on the enzyme surface & impair the enzyme function with strong affinity for the inhibitor to bind and prevent the catalysis possibly due to in the enzyme conformation. Ex: heavy metal ions Ag+2,Pb+2,Hg+2
E------- SH +Hg+2 E-S---------Hg+2 + H+
Ex. Hg+2 heavy metal ion as non competitive inhibitor.


c. Un-competitive inhibition: Reversible inhibition, not very common ,the inhibitor doesn't bind with the enzyme but only bind with enzyme-substrate complex
E + S ES E+P
+
I
ES
I



2.Irreversable Inhibition:The inhibitors bind covalently with the enzymes and inactivate them which is irreversible and the inhibition is usually toxic substance.Ex:Di isopropyl flurophosphate(DFP) is a nerve gas irreversibly bind with enzyme at the active site.هو غاز الاعصاب اكتشف من قبل الالمان في الحرب العالمية الثانية Organophosphorous compound(insecticides) block the activity of Ach.esterase essential for nerve conduction resulting in paralysis of vital body function.
Diagnostic importance of Enzymes: Measurement of enzyme activities in biological fluid (plasma/serum) is of great clinical importance. Enzymes in the circulation is divided into two groups: 1/ plasma specific or plasma functional enzymes: Certain enzymes are normally present in the plasma and they have specific function.


Generally these enzymes activities are higher in plasma than in tissues. They are mostly synthesized in the liver and enter the circulation. Ex: lipoprotein lipase plasmin, choline esterase. Impairment of liver function or genetic disorders leads to fall in the activities of plasma function enzyme.
2/Non –plasma specific: these enzymes are either totally absent or present at a low concentration in plasma compared to their level found in the tissues.All GIT enzymes ,amylase, pepsin,trypsin present in the plasma are known as secretary enzymes.Measurement of the activities of non plasma specific is important in the diagnosis and prognosis of several diseases