4. COENZYMES Many enzymes require for their action on substrate,specific,heat stable,low M. Wt. and organic substens called coenzymes Enzyme which requires a coenzyme for its catalytic action is called apoenzyme and complete catalytic unit which contain enzyme and its coenzyme is called holoenzyme. Coenzyme it self may covalantly or non covalantly bound to enzyme and when coe is covalent linked to its enzyme it will be then called PROSTHETIC GROUP. GROUP. Majority of enzyme in body required coe in their action. Usually classes of enzyme which need coe in their catalytic action are class no 1,2,5,6 however classes 3,4 are usually not required coe in their catalytic action.

5. COENZYMES Many enzymes require for their action on substrate,specific,heat stable,low M. Wt. and organic substens called coenzymes Enzyme which requires a coenzyme for its catalytic action is called apoenzyme and complete catalytic unit which contain enzyme and its coenzyme is called holoenzyme. Coenzyme it self may covalantly or non covalantly bound to enzyme and when coe is covalent linked to its enzyme it will be then called PROSTHETIC Majority of enzyme in body required coe in their action. Usually classes of enzyme which need coe in their catalytic action are class no 1,2,5,6 however classes 3,4 are usually not required coe in their catalytic action.

6. Mechanism of action or role of coenzyme in enzyme reaction 1. coenzyme act as substrates. second substrate In many reaction which required coenzyme, coenzyme itself may be considered as a second substrate or as acosubstrate behavior that is explained by following two explanation; Ex; - In many reaction which required coenzyme, coenzyme itself may be considered as a second substrate or as acosubstrate behavior that is explained by following two explanation; Ex; - alcohol + NAD aldehyde + NADH + +H + alcohol + NAD aldehyde + NADH + +H + Chemical reaction which occurs in the coenzyme is usually opposite to chemical reaction that occur in the first substrate ex: in the oxidation reduction while the first substrate is oxidized the coenzyme (second substrate) is reduced and also in transamination the first sub. Will lose an amino group while the second sub. Will accept amino group

7. Another example: Chemical reaction which occur in the coe. Which are important in the biochemical and physiological condition Chemical reaction which occur in the coe. Which are important in the biochemical and physiological condition - Ex: The importance of the ability of muscle to convert of pyruvate to lactate,in muscle working anaerobically as in the eq: - Ex: The importance of the ability of muscle to convert of pyruvate to lactate,in muscle working anaerobically as in the eq: Pyruvate + NADH +H Lactate +NAD

8. NAD important for formation of ATP by glycolysis and kreb cycle: NAD important for formation of ATP by glycolysis and kreb cycle: Importance of this reaction is not in conversion of pyruvate to lactate but in the formation of oxidized NAD because this NAD will be linked to a series of reaction will result in formation of ATP which will permit a continue of muscle working and exercises and also whiteout NAD glycolysis cannot continue. Importance of this reaction is not in conversion of pyruvate to lactate but in the formation of oxidized NAD because this NAD will be linked to a series of reaction will result in formation of ATP which will permit a continue of muscle working and exercises and also whiteout NAD glycolysis cannot continue.

9. II-Action of coe as group transfer element in the intermediary metabolism: In this aspect the coe will act as an intermediate reagent in transferring a group G ‎From adonar molecule to an acceptor molecule and in this coe. May act an acceptor mole. ( redox reaction ) or it may act as an intermediate compound between a donor and acceptor compound. G –D COE A-G D COE- G A

10. According to the nature of group transfer mechanism the coenzyme are classified into two main group. H+H+H+H+ I- H+ Transfer COENZYME includes: NAD+,NADP+, FAD, FMN, COEQ, and Lipoic 1-acid. II-NON H+ Transfer COENZYME include phosphosugar (ATP and ADP), COEA (COA-SH)C, thiamin pyrophosphate, pyridoxal phosphate (B6),Biotin, B12, Foliate and lipoic acid.

11. Enzyme Specificity The most significant properties in the enzyme catalytic reaction is the ability of the enzyme in catalyze one specific reaction and no other that is a characteristic of enzyme and when these enzyme is absent the respective reaction will not occur and this behavior is called specificity of enzyme and this behavior is usually appear in the following TWO properties.

12. I-optical specificity: The enzyme has an absolute specificity in particular optical region of the substrate.ex: with exception of enzyme epimerase (racemes) which catalyze interconvertion between optical isomerase. Almost all human enzyme are being specific for an optical part of substrate. ex: enzyme acting on CHO. Metabolism (sugar breakdown )are usually specific for D-sugar not act on L-sugar or other enzyme acting on amino acid metabolism are usually acting on L- amino acid (not D- amino acid ) with exception of D- amino acid oxidase in the kidney

13. II- Selective group: I n this properties enzyme is usually affective on specific chemical group that is present in the structure of substrate. ex: glycosides glycoside glycosides catalyze hydrolysis o f glycosidic bond between sugar and alcohol are highly specific for sugar portion not specific for alcohol. Esterase ester

14. Trypsin and pepsin act on peptide bond. Some enzymes have a higher degree of specificity ex: amino peptidase act on amino group, carboxypeptidase act on carboxy end of peptide bond. Chymotrypsin will act on peptide bond on which carboxy terminal end of peptide bond is being contributed to an aromatic a.a. Which may be phenyl alanen, tyrosine and tryptophane split of a.a one at atime from the carboxy or amino terminal end of polypeptide chain respectively.

15. Lastly there are certain metabolic process in body,. Ex: metabolic processes which are involve in biosynthesis process usually tend to use NADP, NADPH as a coenzyme ex: in cholesterol and steroid biosynthesis where as breakdown process or catabolic process usually tend to use NAD. -HOCH2 CHNH2COOH Tyrosine

16. Measurement of enzyme activity S +E ES P +E Enzyme present in body tissues in very small amount that many methods are not so sensitive to measure their concentration and so indirectly enzyme activity can be measure by either measuring the rate of decrease in the substrate concentration or the rate of increase in product formation and for this purpose different units were used for expressing enzyme activity such as King- Armstrong unit (K.A.U.) For measurement the activity of alkaline and acid phosphates.

17. Smoggy unit to measure Amylase activity. Smoggy unit to measure Amylase activity. Activity of enzyme which define by the (I.U) which is defined as the enzyme activity which is responsible for the decrease in one micromole of substrate or increase of one micromole of product per minute. Activity of enzyme which define by the (I.U) which is defined as the enzyme activity which is responsible for the decrease in one micromole of substrate or increase of one micromole of product per minute. Katal unit is the rate of micromol of decrease in substrate or micromole increase in product per second. Katal unit is the rate of micromol of decrease in substrate or micromole increase in product per second..

18. Enzyme Kinetic Include the chemical changes, which occur in the enzyme catalyst reaction and the factors affecting enzyme activity. Include the chemical changes, which occur in the enzyme catalyst reaction and the factors affecting enzyme activity. Catalytic Site: The large size of the enzyme molecule in comparison with substrate size that a small part or limited number of amino acids in the enzyme molecule is being responsible for the catalytic reaction these size is called CATALYTIC SITE or ACTIVE SITE or ACTIVE CENTER of the enzyme. There are two theory or model to explain the interaction between the substrate and enzyme. The large size of the enzyme molecule in comparison with substrate size that a small part or limited number of amino acids in the enzyme molecule is being responsible for the catalytic reaction these size is called CATALYTIC SITE or ACTIVE SITE or ACTIVE CENTER of the enzyme. There are two theory or model to explain the interaction between the substrate and enzyme.

19. I-Lock and key or rigid template model theory : According to enzyme catalytic reaction the active site of the enzyme by itself is complementary in shape to that of the substrate this theory has been discovered by Emil Fischer and is still useful for understanding certain properties of enzyme. This theory has a disadvantage the substrate-binding site has to be reshaped to the size or to the shape of the enzyme.

20. II-Induced fit model theory This theory discovered by Koshland in which there is a source of flexibility in substrate – enzyme binding in which certain physical changes take place in the enzyme that include arrangement of certain a.as both to the substrate binding site and at catalytic site. These changes are called (conformational changes) and the site in which these changes take place are called Allosteric site being important for the enzyme catalytic reaction. This theory is more flexible thane the template theory and it has wide application in explaining the mechanism of the majority of enzyme – catalytic reaction.

22. Factors affecting enzyme activity There are six major factors which affect enzyme catalyst reaction : There are six major factors which affect enzyme catalyst reaction :TemperaturepH Enzyme Concentration Substrate Concentration InhibitorsActivators

23. I-Temperature: Over limited rang of temp. there is usually an increase in enzyme activity with increase of temp. of reaction until most rapid reaction will occur at certain temp. Beyond this temp. a decline in enzyme activity will occur. Temp. at which most rapid reaction will occur is called optimum temperature. Over limited rang of temp. there is usually an increase in enzyme activity with increase of temp. of reaction until most rapid reaction will occur at certain temp. Beyond this temp. a decline in enzyme activity will occur. Temp. at which most rapid reaction will occur is called optimum temperature.

24. (Q 10 =2 ): Is called temp coefficient. Is defined the velocity of many biological reaction is a doubles with 10 c* rise in temp. (Q =2) and halved if the temp. is decreased by 10 c*. Optimum temp. 37-40 c* Activity of Enzyme Temp

28. II- pH For many enzyme in the body working pH is usually between 5-9 but there are certain exception e.g.. pepsin,tyrosine,and acid phosphates act in PH less than 5 and alkaline phosphates act in pH act in more than pH 9. For many enzyme in the body working pH is usually between 5-9 but there are certain exception e.g.. pepsin,tyrosine,and acid phosphates act in PH less than 5 and alkaline phosphates act in pH act in more than pH 9. Optimum pH for any enzyme is defined as pH at which enzyme activity is more rapid Optimum pH for any enzyme is defined as pH at which enzyme activity is more rapid Optimum pH of some enzymes : Pepsin the pH is 1. 5 Trypsin the pH is 7.7 E PH

30. III-Enzyme concentration [ E ]: There is a direct proportional relation between rate of enzyme catalyzed reaction and enzyme concentration.

32. IV-Substrate concentration: [ S ]

33. Km Km E 100% 50%

34. Km Km : The Michaelis menten constant. Km : The Michaelis menten constant. The quantitative relationship between substrate concentration and Vmax. For different enzymes, it is defined as that substrate conc. at which a given enzyme give one – half it maximum velocity. In many cases the Km is an inverse measure of the affinity of the enzyme for its substrate : the lower the Km the higher the affinity. The quantitative relationship between substrate concentration and Vmax. For different enzymes, it is defined as that substrate conc. at which a given enzyme give one – half it maximum velocity. In many cases the Km is an inverse measure of the affinity of the enzyme for its substrate : the lower the Km the higher the affinity. Vmax [S] Vmax [S] Km+[ S] Vi=

35. Inhibitors: ( - ve modulators ) Are substances, which cause inhibition of enzyme activity or decrease the velocity of the enzyme catalyst reaction.inh. are classified into two classes: 1-Competitive inhibitors: Are substances usually have similar in the structure with the substrate and therefore they may be called substrate analogue and so there will be competition between substrate and inh. For binding with the substrate binding site of the enzyme.In the presence of competitive inh. change in enzyme kinetic will cause increase in Km will v-max stay constant.

36. KM Ethylene glycol ( anti freeze) Itself is not toxic, rather the harm is done by oxalic acid, an oxidation product of ethylene glylycol. the first step is the oxidation of ethylene glycol by alcohol dehydrogenase. This reaction can be effectively inhibited by the administration of nearly intoxicating dose of ethanol.the basis of this reaction is that ethanol act as competitive inhibitor of oxidation of ethylene glycol to adehyde product. The same reaction is used in therapy of methanol The same reaction is used in therapy of Methanol poisoning. CH2OH—CH2OH---  CHO—CH2OH---  COOH—COOH oxalic acid (toxic)alcohol dehydrogenase inhibited by ethanol Ex: Treatment of ethylene glycol poisoning by competitive inhibitors.

38. II-non- competitive inhibitors: These inh. have similar or no similar in the structure with substrate and therefore they will not compete with the substrate binding sites these inh. bound to the allosteric sites of the enzyme which are unaffected by substrate but they are importuned in showing the catalytic effect of the enzyme. These type of inh. still can show change in enzyme kinetic which include 1-km remain constant. II-decrease in v-max.ex:

39. Many drugs useful in medicine appear to function because they can inhibits or poisoned certain enzyme in tissues the compound (nerve gas) diisopropyl fluorophosphat DFP. which inhibits the enzyme choline esterase.Choline esterase catalyze the reaction which take place at the junction between certain cell in the nervous system or an enzyme play important role in transmission of nerve impulse.DFP combines with the hydroxyl group of serine residue in the active site of the choline esterase molecule. F  OH-CH2-E (Choline esterase) (CH3) 2 CHO-P-O-CH(CH3) 2 DFP O Many drugs useful in medicine appear to function because they can inhibits or poisoned certain enzyme in tissues the compound (nerve gas) diisopropyl fluorophosphat DFP. which inhibits the enzyme choline esterase.Choline esterase catalyze the reaction which take place at the junction between certain cell in the nervous system or an enzyme play important role in transmission of nerve impulse.DFP combines with the hydroxyl group of serine residue in the active site of the choline esterase molecule. F  OH-CH2-E (Choline esterase) (CH3) 2 CHO-P-O-CH(CH3) 2 DFP O

40. Non competitive inh. Include inhibition of many metabolic process in the body ex: poisoning by heavy metals such as mercury Hg and lead pb,cd, (irreversible comp. Inh. )also poising by cyanid,cyanide which combine reversibly with the iron atom of some iron contain enzyme to yield an inactive form. Activators (+ ve modulators or cofactor ). Ex: activation of lipase by the metal Ca. In this reaction lipase hydrolyze the substrate ( triglyceride into F.A ) will cause inhibition the action of lipase, but by adding Ca + Triglyceride lipase F.F.A. +Ca+2 Ca-F.F.A.complex Lipase -ve inhibit activity of the E lipase

41. this metal Ca will form a complex with product ( F.F.A) lead to the formation of Ca F. F.A Complex and this new product has no inhibitory effect on lipase. Role of selective proteolysis in creation catalytic sites of the enzyme. Many proteins in body including enzymes are manufactured in the body and secreted in an inactive form,but following selective proteolysis process ( cleavage or clips process ) the active form of the protein will appear, where the protein is enzyme, its proportion form is called Proenzyme.

42. Pro/protein protein Cleavage Proenzyme or zymogen enzyme Prohormone hormone e.g. Inactive proteins in the body which are formed or manufactured in proportion forms : 1.Hormones : insulin,glucagon 2.Enzymes : all digestive enzymes lick tyrosine, pepsin, and chymotrypsin 3.Blood clot formation 4.Connective tissue protein: collagen. During physiological need these inactive form convert to the active one.

43. Isoenzyme These means enzyme multiplicity that is the presence of the some enzyme in multiple form. These multiple form have the same catalytic properties but they are different in their physical properties ex: chorine esterase present in the erythrocyte and nerves tissue is different form the enzyme present in plasma and liver. There are six mechanisms that are responsible for appearance of isoenzyme: 1-genetically distinct or differ protein : which include by this mechanism each isoenzyme of the original enzyme is being genetically differ protein or amino acid composition and sequence from other isoenzyme of the same enzyme ex : of this mechanism include isoenzyme present in mitochondria and cytosolic isoenzyme like Asparatate transaminase (AST ) and Glutamate oxaloacetate transaminase

44. 2- Differences in subunit structure : Some enzyme may contain more than one structural subunit e.g Creation Kinase (CK) consist of two subunit called dimer either M or B, M because present in muscle while B present in brain. so CK can form three different isoenzyme from the possible combination of the dimer leading to isoenzyme or CK1 (BB) Which present mainly in the brain, CK2 (MB ) That is present in cardiac muscle CK3 (MM) which is present in skeletal muscle.anther e.g. LDH enzyme is tetrameter formed of 4 structural subunit, which are two kinds of polypeptide chains either H subunit or M subunit which can form 5 types of tetrameter

45. H H H H (H 4) Present in the heart and erythrocyte. LDH 1 α1 28 (15-30) H H H M (H 3 M ) LDH 2 α 2 36 (22-50) H H M M ( H 2 M 2 ) LDH 3 β 23(15-30) H M M M ( H M 3 ) LDH4 γ 6 M M M M ( M 4 ) LDH 5 γ 2 6 (0-15 ) elevated in acute hepatitis,acute muscle injury,muscular dystrophies.

46. Thus skeletal muscle is rich in isoenzyme that has the highest affinity for pyruvate and conversion it most rapidly to lactate ( Km is low ). 3- Isoenzyme due to allelic variation : enzyme might appear in more than in one form due to ( a point of mutation ) that include replacement of one amino acid sequence by another amino acid that lead to the appearance of enzyme in more than one isoenzyme ex: glucose –6- phosphate dehydrogenase which is present in two form : A form, B form A form ( contain Aspartic acid ) B form ( contain Aspargin ) 1.4-combination with charged group : ex : Alkaline phosphates is present in multiple form -(isoenzyme ) due to the different in combination of these isoenzyme with charged group. Present in liver, bone, intestine, placenta.

47. 1. 5- Isomerism : some enzyme may appear in more than one form due to the formation of different isomers which may be positional or geometric isomers choline esterase present in more than one isoenzyme, present in nerve tissue, liver and erythrocyte, malate dehydrogenase ( MDH ) 2. 6- Polymerization : due to different in the aggregation of different amino acid ex: choline esterase.