Part I MCQs (One Best Type)

1. Pakistan Society Of Chemical PathologistsDistance Learning Programme In Chemical Pathology(DLP-2)Lesson No 9Clinical EnzymologyBy Brig Aamir IjazMCPS, FCPS, FRCP (Edin)Professor of Pathology / Consultant Chemical PathologistAFIP Rawalpindi

2. Part IMCQs (One Best Type)

3. b. Dual specificity Q.1: Xanthine oxidase is an important enzyme of purine metabolism. In which category of enzymes you will put this enzyme in terms of enzyme specificity: a. Absolute specificityb. Dual specificityc. Group specificityd. Linkage specificitye. Stereo-chemical specificity

4. Chemical Specificity of Enzymes

5. b. Cardiac troponin Q 2.Enzymes also show tissue specificity of variable degree. Which of the following enzymes (or biomarkers) is the most tissue specific:a. ALTb. Cardiac troponinc. γ glutamyltransferase (γ GT) d. Lipasee. α amylase

6. Cardiac troponins (both I and T) are most tissue specific as they are genetically distinct from their skeletal counterparts • Lipase is not specific to pancreas. • Lipase in serum is mostly from pancreas but it can be from gastric, pulmonary and intestinal mucosa Tissue Specificity of Enzymes

7. b. ALT Q 3: Lymphatic drainage is one of the mechanisms used by enzymes to reach blood circulation after they are released from diseased organs. In which of the following enzymes (or biomarkers) lymphatic drainage is the least important route of reaching blood circulation once it is released from its most specific organ:a. Aldolaseb. ALTc. Amylased. Cardiac Troponin Te. CK-MM.

8. Most enzymes reach blood circulation through lymphatics • Muscular enzymes use this route to the greatest extent but intestinal, pancreatic and myocardial enzymes also use this route • Liver is very vascular tissue with highly permeable capillaries. So liver enzymes reach circulation by direct transfer. Lymphatic Drainage as Important Route of Entry in Circulation

9. e. γ GT Q 4: Enzymes are released as a result of tissue injury or damage. But higher serum concentrations of which of the following enzymes may be found without any damage to its organ of origin:a. ALTb. ASTc. CKd. LDe. γ GT

10. Some enzymes are increased without a damage or injury to the organ • Increased ALP in growing bones and bone diseases • γ GT, ALP and 5 Nucleotidase increase due to their increased production as a result of stress on the liver – process called enzyme induction • γ GT increases in alcoholism even prior to liver damage. • Some drugs and toxic substances can also cause enzyme induction. Increased Enzyme Production without Injury

11. b. Amylase Q 5 :Clearance of enzymes from circulation depends on several processes. Which of the following enzymes is least dependent on liver for its clearance:a. Adenylate kinaseb. Amylasec. ASTd. CKe. Intestinal fraction of ALP

12. Mostly enzymes are cleared from circulation by a receptor- mediated process called ‘endocytosis’ • This endocytosis of the enzymes takes place mainly in the RE system e.g. Kupffer cells of the liver. • Metabolic break-down is now considered not an important mechanism of enzyme clearance • Intestinal fraction of ALP is an example of using Hepatic endocytosis as a clearing mechanism Enzyme Clearance

13. Alpha Amylase is an example of small-sized enzyme which is excreted through the kidneys. • It can be falsely increased in CKD and ARI and should be interpreted carefully if acute pancreatitis is to be excluded in these patients • In such cases we have to use GFR (creatinine clearance) – Amylase index for interpretation of results Renal Clearance of Enzymes

14. Can you name an enzyme which is also very small in size and which filters through glomeruli but completely reabsorbed in tubules – so not found in urine???? • For answer join Skype session of Friday or Saturday A Chocolate (Virtual) Question!!

15. a. Allozymes Q 6 :The sub-types of enzymes which result due to individual genetic variation following Mandalian laws are called:a. Allozymesb. Homopolymericisoenzymesc. Hybrid isoenzymesd. Isoformse. True isoenzymes

16. Isoenzymes are genetically distinct sub-types of enzymes while isoforms are due to post-translation modifications in enzymes • Example: • CK-MM, CK-MB and CK-BB are isoenzymes of CK (now called CK1, CK2 and CK3 respectively). • CK-MB1 CK-MB2, CK-MB3 are isoforms of CK-MB Isoenzymes and Isoforms

17. Isozymes, aredifferent forms of the same enzyme encoded at different gene loci • Alloenzymes: allozymesare encoded by different alleles at an enzyme gene locus. These are due to mutations causing differences in basic and acidic amino acid composition, but no change in enzyme function Example: More than 400 mutations in a locus on X Chromosome causing 400 Allozymes of G-6-PD Isozymes and Allozymes

18. c. Katal Q 7 : The SI unit for enzyme activity measurement is:a. µmol/Lb. g/Lc. Katald. mmol/Le. U/L

19. The unit for reporting enzyme concentration is International Unit/liter • 1 U/L= µmoles of substrate used up per minute of incubation per liter • Katal = moles of substrate used up per second of incubation per liter • 1 U/L = 16.6 nanokatal Units of Enzyme Activity

20. Using data when the reaction is in zero order: 1. calculate change in absorbance per minute 2. correct for dilution factor and serum volume 3. use molar absorptivity to convert from absorbance to micromoles Enzyme Activity and Concentration

21. LD reaction which produces NADH • Molar absorptivity of NADH at 340 nm = 6220 Abs per mol/L (in 1 cm cuvet) • Assume that 10 mL of serum is added to 1 mL of substrate, and Absorbance readings are recorded every 10 seconds during a one minute incubation. 1. calculate Delta Abs for each time interval 2. calculate average delta Abs during 0 order 4. apply formula Example of Calculating Enzyme Activity in Traditional Units

22. d. L-Alaninine; 2-oxoglutarate aminotransferase Q 8 :Enzyme Commission has given Systemic Names to the enzymes. Which one of the following is a Systemic Name of the enzyme and NOT a Trivial Name:a. Acetylcholinestraseb. Aspartate aminotransferasec. Glutamate dehydrogenased. L-Alaninine; 2-oxoglutarate aminotransferasee. γ-glutamyltransferase

23. d. Two substrate reaction when one is water Q 9 :Two or more substrates are commonly used in Clinical Enzymology. Which of the following two-substrate reactions follows one-substrate kinetics? a. Indicator reactionb. Consecutive enzyme reactionc. Ping pong bi bi reactiond. Two substrate reaction when one is watere. Ternary complex reaction

24. Cleland Nomenclature for Enzymes • Cleland has devised a standardized way of referring to bisubstrate (Bi-Bi) enzymatic reactions, which make up 60% of all enzymatic transformations. The substrates, products and stable enzyme forms are denoted as follows: • Substrates are lettered A, B, C and D, in the order that they are added to the enzyme • Products are lettered P, Q, R and S, in the order that they leave the surface of the enzyme • Stable enzyme forms are lettered E, F and G, in the order that they occur • The number of reactants in the reaction are designated by the terms Uni, Bi, Ter and Quad These are transfer reactions so can be presented as • AX + B BX + A Kinetics of multi substrate enzyme catalysed reactions

25. Sequential bi-bi • The first important type of bi-bi reaction is known as sequential, which means that all substrates must add to the enzyme before any reaction takes place • The sequential bi-bi can be • random, any substrate can bind first to the enzyme and any product can leave first • ordered, meaning that the substrates add to and products leave the enzyme in a specific order • A ternary complex (E + both substrates) is formed in both cases

26. BX A AX E.AX E-BX E.AX.B E.A.BX E.A AX BX A Sequential bi-bi

27. Ping-pong bi-bi(double-displacement) • One substrate bind first to the enzyme followed by product P release • Typically, product P is a fragment of the original substrate A • The rest of the substrate is covalently attached to the enzyme E, which we now designate as F • Now the second reactant, B, binds and reacts with the enzyme to form a covalent adduct with the covalent fragment of A still attached to the enzyme to form product Q • This is now released and the enzyme is restored to its initial form, E

28. d. Polymerase Q 10 :Temperature is an important factor controlling rate of enzyme reaction (as we will discuss in Q 11). .Generally enzymes get denatured above 600 C but which of the following enzymes do not get denatured even at 900 C:a. 5 Nucleotidaseb. Amylasec. Nucleased. Polymerasee. Protease

29. Part IIShort Answer Questions:

30. Q. 11: Many factors effect enzyme-catalysed reactions in human body. Please write THREE lines for each of the following factors with ONE example (5 marks):a. Coenzymesb. Competitive inhibitors of enzymesc. Optimal temperature of enzyme activityd. Prosthetic group e. Role of buffers in enzyme reaction

31. Q. 11 a.Co-Enzymmes Slides Courtesy DrSobia AFIP Rwp

32. A diffusible, heat stable substance of low molecular weight that, when combined with an inactive protein (apoenzyme) forms an active compound or complete enzyme called a holoenzyme. Nature of Coenzyme

33. Coenzymes are bound only momentarily to the enzyme during the course of reaction • No reaction takes place unless the appropriate coenzyme is present in the solution • They are smaller molecules than the enzyme proteins themselves Actions of Coenzyme

34. Coenzymes often function as intermediate carriers of electrons, specific atoms or functional groups that are transferred in the overall reaction • These are "helper molecules" that assist in biochemical transformations. • Examples:role of NAD in the transfer of electrons in certain coupled oxidation reduction reactions. • A number of the water-soluble vitamins such as vitamins B1, B2 and B6 serve as coenzymes. Examples of Coenzymes

35. Q. 11 a.Competitive inhibitors of enzymes Slides Courtesy of Slides Courtesy DrMajid AFIP Rwp and Dr Sara Reza QAMC Bwp

36. A competitive inhibitor is usually structural analogue of the substrate that can combine with the free enzyme in such a way that it competes with the normal substrate for binding at the active site • The actual rate of reaction is strictly dependant on the relative concentrations of substrate and inhibitor Competitive Inhibitors of Enzymes

37. Competitive Inhibitors of Enzymes

38. Competitive inhibition is responsible for the inhibition of some enzymes by excess substrate because of competition between substrate molecules for a single binding site. • In two substrate reactions high concentrations of the second substrate may compete with binding of the first substrate. Types of Competitive Inhibition

39. a. AST is inhibited by excess concentrations of substrate 2-oxoglutarate and the inhibition is ccompetitive with respect to L-aspartate. Therefore to maintain a given velocity at high 2-oxoglutrate concentrations, the concentration of L-aspartate has to be increased above the value needed at lower concentrations of 2-oxoglutarate. b. Benzamidinecompetes with arginine for binding to trypsin Examples of Competitive Inhibition

40. Q. 11 c.Optimal temperature of enzyme activity Slides Courtesy DrUzma AFIP Rwp

41. Optimal temperature is the temperature at which an enzyme attains its maximal activity. • Rise in temperature from low temperature to optimum temperature causes an increase in the rate of reaction by increasing the initial energy of substrate leading a decrease in the activation energy and lower the energy barrier of the reaction . Optimal temperature of enzyme activity

42. Optimal temperature is usually around human body temperature i.e. 37oC so currently most of the analytical systems operate at 37oC • Accurate temperature control to within +0.1oC during the enzymatic reaction is essential • The rise in temperature above the optimal temperature leads to decrease in the rate of enzyme activity due to denaturation of enzyme. Optimal temperature of human enzymes

43. Q. 11 d.Prosthetic group Slides Courtesy DrAmina AFIP Rwp

44. Organic co-factors are usually divided into coenzyme and prosthetic group. • Prosthetic group is the non-protein, organic or inorganic part of a holoenzyme. • The active holoenzyme results from the combination of the inactive apoenzyme with the prosthetic group; which in contrast to co-enzyme is tightly bound to the enzyme. Prosthetic group

45. 1.Pyridoxal-5’-phosphate (P-5’-P) in ALT analysis. 2. Heme in haemoglobin. In ALT analysis; incubation period is required for P-5’-P to fully activate apo-aminotranferase to be converted to active holoenzyme. Examples of Prosthatic Groups

46. Q. 11 e.Role of buffers in enzymatic reaction Slides Courtesy DrQurat-Ul-Ain AFIP Rwp

47. Buffers are used in enzyme reaction to maintain optimum pH required to have maximum catalytic activity of enzyme. Optimum pH for in vitro enzymatic reactions • For most of the plasma enzymes(e.g. ALT, AST): 7-8 • For ALP: 10.5 • For Pepsin: 1.5 Role of buffers in enzymatic reaction

48. Rate of enzymatic activity is decreased as pH is changed from optimum one due to following • Ionization of substrate or enzyme or both • Dissociation of key amino acid side chains in the enzyme molecule • Change in three dimensional conformation of the protein resulting in denaturation of enzyme Effects of change in pH on enzymatic reaction

49. pKa value within one pH unit of desired pH of assay to have maximum buffering capacity • Ability to neutralize the effect of • Specimen addition as serum itself is a powerful buffer • Acids or bases formed during the reaction • No interaction with components of assay system • Examples In IFCC reference methods • For ALP - AMP(1-amino 2-methyl propanol) buffer to maintain pH at10.5 • For ALT - Tris buffer to maintain pH at 7.15 Properties of appropriate buffer

50. Q. 12: A scientist is working on development of a new chemical reaction for kinetic estimation of an enzyme in the serum. Please answer following questions in this regard:a. Please clarify whether it will be a Zero-order reaction or First order? b. Which point of the curve he will select for this reaction (‘A’ or ‘B’ in the MichaelisMenten Plot given below). c. While setting up this method what THREE steps are necessary to develop a valid reaction? d. How will he calculate the amount of substrate required for the reaction? e. What step he can add in his experiment to ensure that the reaction proceeds only in forward direction as given in the following equation: E + S ES P + E