1. Enzymes and heart attacks

2. Enzymes: “Helper” Protein molecules

3. Flow of energy through life • Life is built on chemical reactions

4. + + Chemical reactions of life • Processes of life • building molecules • synthesis • breaking down molecules • digestion

5. enzyme + enzyme + Nothing works without enzymes! • How important are enzymes? • all chemical reactions in living organisms require enzymes to work • building molecules • synthesis enzymes • breaking down molecules • digestive enzymes • enzymes speed up reactions • “catalysts” We can’t live without enzymes!

6. + enzyme enzyme + Examples • synthesis • digestion

7. Enzymes are proteins • Each enzyme is the specific helper to a specific reaction • each enzyme needs to be the right shape for the job • enzymes are named for the reaction they help • sucrase breaks down sucrose • proteases breakdown proteins • lipases breakdown lipids • DNA polymerase builds DNA Oh, I get it! They end in -ase

8. active site Enzymes aren’t used up • Enzymes are not changed by the reaction • used only temporarily • re-used again for the same reaction with other molecules • very little enzyme needed to help in many reactions substrate product enzyme

9. It’s shape that matters! • Lock & Key model • shape of protein allows enzyme & substrate to fit • specific enzyme for each specific reaction

10. 2 1 3

11. Enzyme vocabulary • Enzyme • helper protein molecule • Substrate • molecule that enzymes work on • Products • what the enzyme helps produce from the reaction • Active site • part of enzyme that substrate molecule fits into

12. What affects enzyme action • Correct protein structure • correct order of amino acids • why? enzyme has to be right shape • Temperature • why? enzyme has to be right shape • pH (acids & bases) • why? enzyme has to be right shape

13. Order of amino acids • Wrong order = wrong shape = can’t do its job! foldedprotein chain ofamino acids DNA right shape! foldedprotein chain ofamino acids wrong shape! DNA

14. Temperature • Effect on rates of enzyme activity • Optimum temperature • greatest number of collisions between enzyme & substrate • human enzymes • 35°- 40°C (body temp = 37°C) • Raise temperature (boiling) • denature protein = unfold = lose shape • Lower temperature T° • molecules move slower • fewer collisions between enzyme & substrate

15. 37° Temperature humanenzymes What’s happening here?! reaction rate temperature

16. How do cold-blooded creatures do it?

17. pH • Effect on rates of enzyme activity • changes in pH changes protein shape • most human enzymes = pH 6-8 • depends on where in body • pepsin (stomach) = pH 3 • trypsin (small intestines) = pH 8

18. pH stomachpepsin intestinestrypsin What’s happening here?! reaction rate 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 pH

19. SHAPE! For enzymes…What matters?

20. Let’s build some Enzyme Models!

21. Myocardial infarction • Acute myocardial infarction is the rapid development of myocardial necrosis caused by a critical imbalance between the oxygen supply and demand of the myocardium. • 500,000-700,000 deaths inthe US annually.

22. Symptoms Angina pectoralis Dyspnea Nausea and/or abdominal pain Anxiety Lightheadedness and syncope Cough Nausea and vomiting Diaphoresis One problem - Differential diagnosis Pericarditis Aortic Dissection Cholecystitis and Cholelithiasis Laryngeal spasm Anxiety attack and on and on and on… One solution – “Cardiac enzymes” Myocardial infarction

23. Enzymes • Definition: Biological catalysis • Qualities • Efficient • Specific • Stereo-specific - they can tell the difference between isomers • Regulated • Saturable • Inhibitable • Substrate versus product

24. Types of enzymes • All enzymes end in the suffix “_______ase” • Different versions of the same enzyme (often made by alternative splicing) are called isoenzymes or isozymes • General classes of enzymes • Polymerases – nucleic acid synthesis • Transferases – transfer a functional group • Hydrolases – hydrolytic cleavage • Proteases – hydrolytic cleavage of protein chains • Kinases – add phosphate groups to compounds • … and many, many more…

25. Mechanism • Enzymes work by lowering activation energy • If you don’t understand free energy changes, see Box 5A in your book • ∆G is a measure of the ability of a reaction to go forward, but not necessarily the rate • EA is the activation energy. • The rate at which a reaction proceeds is directly proportional to the number of molecules reaching the transition state - that is, those that reach EA. 

26. Things for optimal activity • pH – alters enzyme structure by altering charge • Temperature – increases activity by moving molecules closer to the activation energy, and by making ∆G slightly more negative… until the enzyme "denatures" • Coenzymes – like biotin in amino group transfer – bind reversibly but participate directly • Metal ions – like magnesium in some ATPases.

27. Michaelis-Menten Kinetics • Shows saturation at high substrate concentrations • Vmax – rate at saturation for a given enzyme concentration in moles per unit time • Km – Michaelis constant – substrate concentration that gives ½ maximal velocity

28. How do you measure this crap? • Things you need: • The enzyme • The substrate • A way of measuring either the disappearance of substrate, or the appearance of product, usually photometrically.

29. Other commonly reported values • Turnover • rate at saturation for 1 enzyme molecule (reactions catalyzed per second per molecule) • “Units” • are defined by convention, but are something of an industry standard.  For example… • “One unit of creatine kinase is defined as the amount necessary to catalyze the conversion of one micromole of creatine to creatine phosphate per minute at 25°C and pH 8.9.”

30. Competitive inhibitors • Many drugs (like Cipro and anti-HIV drugs) are enzyme inhibitors • Two major kinds of inhibitors: competitive and noncompetitive. • Competitive inhibitors bind to the active site of the enzyme. • Alter Km but not Vmax. • What will happen to V ifyou push the substrateconcentration very high?

31. Noncompetitive inhibitors • Noncompetitive inhibitors bind somewhere besides the active site. • They alter the behavior of the enzyme in a manner analogous to allosteric regulation • Alter Vmax. • What will happen to V ifyou push the substrateconcentration very high?

32. Regulation Allosteric regulation • A regulatory molecule binds to a site separate from the active site (like small molecules to repressors in operons) • Induced conformational changes regulate the activity of the enzyme • These enzymes usually have catalytic and regulatorydomains • Can have multiple domainsor subunits for different regulators

33. Regulation Allosteric Cooperativity • One substrate aids or impedes the catalysis of another • Implies multiple catalytic subunits. Covalent modification • Adding/removing groups – like phosphate groups by kinases • Cleaving bonds – converting proenzymes to enzymes - like in the blood clotting cascade Association-dissociation of subunits • One protein binds to another, thereby activating the enzymatic activity of one of them.

34. Creatine kinase • Creatine phosphate acts as a backup for rapid ATP regeneration in active tissues • Creatine phosphate is in energetic equilibrium with ATP • Creatine kinase (CK) catalyzes the transfer of phosphate between creatine and ATP/ADP • Provides rapid regeneration of ATP when ATP is low • Creatine phosphate is regenerated when ATP is abundant ADP ATP CK Cr-P Cr

35. Application: Cardiac enzymes • enzymes released from injured myocardium. • Creatine kinase (CK) is the one usually assayed • If CK is found in the blood stream, this implies that the myocardium may have been damaged • Problems: • Tells you little about the time course or severity • Lets you spot really small infarcts. • What else?

36. Creatine kinase isozymes • The enzyme is dimeric • Two different polypeptide chains (M and B) are differentially expressed in tissues • Combine at random to give three isozymes: • CK-MM (primarily muscle) • CK-MB (hybrid) • CK-BB (primarily brain) • The CK-MB has its highest concentration in heart muscle • CK-MB >5% of total CPK strongly suggests myocardial infarction

37. Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y POSITIVE Substrate Determining CK-MB (mass) / CK (activity) • Total CK activity is determined by a simple enzyme assay (phosphocreatine + ADP  ATP) • CK-MB mass is determined by a two-antibody “sandwich” assay. Y Y Y Y Y Y Y Y Y Y Tagged anti-CK-M anti-CK-B coated tube