BIOCHEMISTRY AS A SUBJECT, ITS TASKS. ENZYMES: STRUCTURE, COMMON PROPERTIES, MECHANISM OF ACTION AND CLASSIFICATION

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1 THE MINISTRY OF PUBLIC HEALTH OF UKRAINE ZAPORIZHZHIA STATE MEDICAL UNIVERSITY BIOCHEMISTRY DEPARTMENT BIOCHEMISTRY AS A SUBJECT, ITS TASKS. ENZYMES: STRUCTURE, COMMON PROPERTIES, MECHANISM OF ACTION AND CLASSIFICATION Produced by Ass.professor Krisanova N.V.,

2 Biochemistry static dynamic functional Analysis of chemical composition of the living organism, composition and structure of essential substances, their properties and function is concerned with Study of various metabolic conversions of substances in plants, animals, humans and other living organisms. Study of energy exchange in cells Study of some chemical processes that constitute a basis of various manifestations of vital activity, for example: 1) a transmission of nervous impulses; 2) muscular contraction, etc.

3 Enzymology A study of catalysts in living species - enzymes Biochemical genetics Researches of genetic information transmission mechanism Bioenergetics: Investigations of energy exchange in cells and its regulation by some factors As a science B. is divided in many directions: f. e. Molecular immunology Biomolecules promoted immunal system function in order to prevent some somathic diseases of humans Clinical Biochemistry is concerned with 1) investigation of some indexes of whole blood, blood plasm, urine, saliva, gastric juice at healthy and diseased people. It is a main helper for medicine 2) methodical developments in diagnosis and treatment of various diseases

4 Enzymes are catalysts of protein nature What proteins? Fibrous or Globular? - Globular, mainly! Simple or Conjugated? Both! But conjugated enzymes are more in a quantity!

5 Creation of active site may be in globular structure only

6 Ribozymes Ribozymes exist in nature as catalytic RNA molecules that either aid the hydrolysis of their own phosphodiester bonds or cause the hydrolysis of bonds in other RNAs. Ribozymes also play a role in other vital reactions such as RNA splicing, transfer RNA biosynthesis, and viral replication. They also catalyze the aminotransferase activity of the ribosome.

7 Non-protein part of conjugated enzyme may be nonorganic or organic compound: Non-organic: Metal ions: Ca 2+, Mg 2+, Zn 2+, Co 2+, K +, Na +, Cu 2+ ; Enzymes: Metalloproteins Phosphoric acid residues: H 2 PO 4-, HPO 4 2-, PO 4 3- ; Enzymes: Phosphoproteins Organic compounds: 1. ATP, AMP, ADP, etc. nucleotides. 2. Carbohydrates such as glucose, galactose, mannose, etc. Enzymes: Glycoproteins 3. Vitamins and their derivatives

8 The use of some vitamins in the structure of enzymes Vitamin B 1 Thiamine B 2 Riboflavin B 5 Pantothenic acid B 3 or PP Nicotinic acid or Nicotin amide Coenzyme or prosthetic group TPP (Thiamine pyrophosphate) FMN (Flavin MonoNucleotide) FAD (Flavin Adenine Dinucleotide) CoASH (Coenzyme A), ACP (Acyl carrier protein) NAD +, NADP + (Nicotinamide Adenine Dinucleotide, Nicotinamide Adenine Dinucleotide Phosphate) Type of the reaction catalyzed by the enzyme Oxidative decarboxylation of keto acids Oxidation-Reduction Oxidative decarboxylation of keto acids Activation of free acids Palmitate synthase complex Oxidation-Reduction Oxidative decarboxylation of keto acids

9 Active site, composition For Simple enzyme : it is composed from amino acid residues Ser, Tyr, Asp, Glu, His, Thr, Cys, mainly For Conjugated enzyme: it is composed from vitamin derivatives or other cofactors, and some amino acid residues, too.

10 A majority of enzymes are synthesized as precursors of enzymes (inactive form, pro-enzyme). There are some ways of activation of pro-enzyme to form active enzyme: Non-complete proteolysis of precursor; Autocatalysis: ability of active form of enzyme to produce itself from pro-enzyme. Allosteric activation of pro-enzyme: 1) linkage of allosteric activator to allosteric site; 2) covalent modification : phosphorylationdephosphorylation

11 CH 3 NADH+H + NAD + CH 3 C O CH OH COOH Lactate DHase COOH Izoenzymes of Lactate dehydrogenase (LDH): LDH1 LDH2 LDH3 LDH4 LDH5 H4 H3M H2M2 HM3 M4 H and M subunits of the quaternary structure of enzyme Myocardium LDH1 and LDH2 Liver LDH3 < LDH4 < LDH5 Skeletal muscle LDH3 > LDH4 > LDH5 Kidneys LDH3 Erythrocyte LDH1

12 Multienzyme system Pyruvate dehydrogenase complex CH 3 (CO)COOH+ CoASH+ NAD + CO 2 +CH 3 CO-CoA+NADH Pyruvate Acetyl-CoA

13 Example of Multienzyme system Pyruvate dehydrogenase complex (organization in space)

14 The behavior of enzyme as catalyst Free energy Initial state (1) (2) Transition state for uncatalyzed reaction (1) Transition state for reaction catalyzed by the enzyme (2) Final state Duration of reaction E activation = E transition state E initial state 14

15 Chemical reaction : S P (1) S P (2) E + S ES ES* EP E + P (1) E + S ES ES* EP E + P (2) E Enzyme S Substrate S* - Intermediate product of the reaction P the final product of the reaction (1) reversible reaction; (2) -irreversible reaction.

16 Illustration of ES complex transformation (case: S P 1 +P 2 )

17 The specificity of enzyme is determined by: The functional groups of the substrate (or product) The physical proximity of these various functional groups The functional groups in active centre of enzyme and its cofactors (coenzymes) ES complex is formed due to interactions of these functional groups. The type of bonds for ES complex formation: Hydrogen bonds Electro-static interactions Covalent bonds Magnetic attractions

18 Specificity subtypes Absolute : one enzyme one substrate, only Relative group : one enzyme a group of substrates, containing the same structural fragment that is transformed by the enzyme Stereochemical : one enzyme one substratestereoisomer to be converted into the product

19 The lock and the key theory (Fisher E., 1930th) The active centre of the enzyme (the lock) is complementary in the conformation to the substrate (the key), so that enzyme and substrate recognize one other one.

20 The induced-fit theory (D.E. Koshland, 1950th) 1.Orientation and approach of an enzyme and a substrate relative each other in space. 2.The enzyme contact with the substrate (ES complex formation) is the induced fit state of the enzyme to the substrate. 3.The attraction provokes conformational changes in the enzyme active site (some strain is there), and there is some deformation in the substrate structure, too. 4.All these changes promote quickly the reaching of the transition state of the reaction.

21 The lock and the key theory (Fisher E., 1930th) The induced-fit theory (D.E. Koshland, 1950th)

22 Enzymes catalyze reactions by utilizing the same general reactions as studied in organic chemistry: Covalent catalysis Metal ion catalysis Catalysis by alignment (approximation) Acid-base catalysis Additional free energy is obtained through the Binding Energy (binding of the substrate to the enzyme.) Binding energy often helps stabilize the transition state, lowering E activation

23 Carbonic Anhydrase contains an important cofactor at the active site, namely a zinc ion, that helps to activate water molecules prior to their reaction with CO 2 : CO 2 + H 2 O HCO 3- + H + Biochemistry 3070 Enzyme Mechanisms 23

24 Carbonic Anhydrase The binding of water to zinc, reduces the pka for water from its normal 15.7 down to 7. This allows the formation of the strong hydroxide (HO - ) nucleophile at neutral ph: Biochemistry 3070 Enzyme Mechanisms 24

25 The enzyme then positions CO 2 for nucleophilic attack by the hydroxide, resulting in the formation of bicarbonate. Water then displaces the product, starting the cycle again. Biochemistry 3070 Enzyme Mechanisms 25

26 Acid base catalysis Some specific amino acid residues in active centre of enzyme may be donors or acceptors of protons during the catalysis (the conversion of a substrate to a product), such as: Donors Acceptors - COOH - COO- - NH + 3 -NH 2 - SH - S - - OH - O -

27 Covalent catalysis. Chymotrypsin Ser-195 attacks substrates, forming an ester linkage to the substrate as the first step in the reaction mechanism. This leaves part of the substrate covalently bonded to the enzyme. Water subsequently enters, deacylating the enzyme by hydrolyzing the ester bond. Biochemistry 3070 Enzyme Mechanisms 27

28 Main factors to regulate activity of enzymes: Substrate concentration Enzyme concentration ph of the medium Temperature of the medium Activators Inhibitors: o Competitive o Non-competitive o Allosteric (feed-back subtype)

29 The influence of the ph on enzyme activity (enzymes of cytoplasm) A,% 100% X L * * * Y I K 7-8 M 0 14 ph

30 The influence of the temperature on enzyme activity A % 100% 0 0 C I I 38 0 C 70 0 C t 0 C

31 Enzyme code translation For example: Tyrosine aminotransferase: E.C Class: Transferase Subclass: the type of the group that is transferred (amine group) the ordinal number of this enzyme Sub-sub-class: the nature of substrate (donor)

32 1.OXIDO REDUCTASE ACTION 1) H 2 C CH 2 H C CH 2H +, 2 ē 2) A +2 - e - B 3) A +O - 2 AO 2 4) SH + O 2 S-OH + H 2 O 2H +, 2 e

33 2. TRANSFERASE ACTION Group transported by the enzyme A-X + B B-X + A Donor Acceptor Two products

34 3. HYDROLASE ACTION A-X + H-OH H-X + A-OH Substrate hydrolyzed by the enzyme Two products 34

35 4. LYASE ACTION 1) CH CH 2 H C CH OH H 2 O H 2 O 2) A-X X + A Substrate distroyed by the enzyme Two products

36 5. ISOMERASE ACTION H 2 C OH O P O H 2 C O-P OH O H 2 C OH OH OH OH OH Glucose-6-P OH Fructose-6-P C 6 H 13 PO 9 ISOMERS C 6 H 13 PO 9 36

37 6. LIGASE ACTION X + A + ATP A-X + ADP + H 3 PO 4 Three substrates Energy source Three products 37

38 Enzymes decrease the energy of activation E activation = E transition state E initial state Free energy (1) Transition state for uncatalyzed reaction (1) (2) Transition state for catalyzed reaction (2) Initial state Final state Duration of reaction

39 E + S K +1 K -1 K +2 ES EP E + P K -2 K +1 the rate constant for the formation of ES K -1 the rate constant for dissociation of ES K +2 the rate constant for dissociation of ES to E plus P. K -2 the rate constant of ES formation from E and P.

40 Initial reaction velocity V = K K 2 1 V K K s ES-complex dissociation constant K m = K s + Michaelis Menten equation V K max m max s Briggs - Haldeine equation Initial reaction velocity V = K m - Michaelis constant [ S] [ S] [ S] [ S]

41 The determination of Km value using graph curve K m equals substrate concentration at which the velocity is half-maximal. The lower the K m the higher the affinity of the substrate to the enzyme

42 Lineweaver-Burk method The reverse values to V and S are produced from Briggs Haldeine equation: 1) 1 Km [ S] 1 Km 1 1 ; 2) V V max [ S] V V S V 1/V max he straight-line graph is obtained by plotting. max 1 V opposite 1 [ S] 1/V max a tga= Km / Vmax - 1/K m 1 / [S]

43 The rate of enzymatic reaction is regulated by enzyme concentration: This dependence is considered only if: ph, t 0 C optimal, [S] >> [S] saturation V 0 [E]

44 The use of Lineweaver-Burk method for the investigation of inhibition type 1/V + I - I Competitive inhibition (reversible type): 1/[S] +I at the presence of inhibitor ; -I inhibitor is absent

45 Succinate dehydrogenase is inhibited by competitive inhibitor malonate OH OH C O C O CH 2 CH 2 C OH O FAD FADH 2 H C C C OH H O HO C O C H 2 Malonate C O OH Succinate Trans-fumarate

46 Non-competitive inhibition (reversible type): 1/V + I -I +I at the presence of inhibitor; -I inhibitor is absent Example: E cytochrome C oxidase I - CN - 1/[S]

47 Activator Inhibitor Schematic representation of allosteric enzyme activity inhibition

48 Irreversible Inhibition This type of inhibitors binds covalently or so tightly to the active centre of enzymes that they are inactivated irreversibly. Affinity labels; Example: Diisopropyl fluorophosphate The reactive group of Inhibitor permanently blocks the active site of the E from the S because the group reacts covalently with amino acid residue. Suicide inhibitors; Example: Allopurinol These are substrate analogs that are transformed by the catalytic action of the enzyme. The product of this reaction is highly reactive and subsequently combines covalently with an amino acid residue in the active centre, thus inactivating the enzyme. Transition state analogs; Example: Penicillin There are substrates analogs do not covalently modify the enzyme but bind the active site so tightly that they irreversible inactivate the E.

49 Total enzyme activity (T.A.) determination: T.A. = -ΔS/Δt; = ΔP/Δt; =ΔCoenzyme/Δt Units: Katal, International Unit (IU) Specific activity (S.A.) is the number of units of total activity per milligram of total protein concentration [C] present in the sample: S.A. = T.A. / [C] This type of activity is used in researching works. Turnover number (TN) is the number of substrate molecules metabolized per one enzyme molecule per unit of time. For example: Carbonic Anhydrase has TN= /min.

50 THANK YOU For ATTENTION! 50