1. MLAB 2401: Clinical Chemistry Keri Brophy-Martinez Enzymes: Overview

2. Enzymes Functional proteins that catalyse biological reactions Involved in all essential body reactions Found in all body tissues – Seen in serum following cellular injury or from degraded cells Decrease the amount of free energy needed to activate a specific reaction

3. General Properties of Enzymes Not altered or consumed during reaction Reusable Accelerate speed of reactions

4. General Properties of Enzymes Holoenzyme – Functional unit – Consists of: Apoenzyme Cofactor/coenzyme Proenzyme/zymogen – Inactive enzyme Holoenzyme

5. General Properties of Enzymes Role – Increase reaction rates while not being consumed or altered Enzyme – Substrate Product

6. Definitions and Related Terms Active site – Specific area of the enzyme structure that participates in the reaction(s)/interacts with the substrate

7. Definitions and Related Terms Allosteric site – Non-active site – May interact with other substances resulting in overall enzyme shape change

8. Definitions and Related Terms Isoenzymes – Structurally different enzymes that catalyze the same reaction Multi molecular form Similar catalytic activity Differing biochemical or immunological characteristics Can detect by different electrophoresis patterns, absorption patterns, or reaction with specific antibodies

9. Definitions and Related Terms Cofactor – Non-protein substances required for normal enzyme activity – Types Activator: inorganic material such as minerals – (Ca 2+, Fe 2+) Co-enzymes: organic in nature – (ATP, ADP, nicotinamide)

10. Enzyme Kinetics Reactions occur spontaneously if energy is available Enzymes lower the activation energy for the chemical reactions

11. Enzyme Kinetics Activation energy – Excess energy that raises all molecules at a certain temperature to the activation state

12. Enzyme Kinetics Basic reaction – S + E ES E + P – Where S= substrate – Substance on which the enzyme acts E= Enzyme ES= enzyme-substrate product – Physical binding of a substrate to the active site of enzyme P= Product

13. Enzyme Kinetics & Specificity Enzymes differ in their ability to react with different substrates – Absolute specificity Enzyme combines with only one substrate and catalyzes one reaction – Group specificity Combine with all substrates containing a specific chemical group – Bond specificity Enzymes specific to certain chemical bonds – Stereoisomerism Enzymes that mainly combine with only one isomer of a particular compound

14. Michaelis-Menten Relationship of the reaction velocity/rate to the substrate concentration The Michealis-Menten Constant (Km) The substrate concentration in moles per liter when the initial velocity is ½ V max. Michaelis-Menten Curve

15. Michaelis-Menten First order kinetics – Rate is directly proportional to substrate concentration Zero order kinetics – Plateau is reached – depends only on enzyme concentration

16. Michaelis-Menten Equation used to distinguish different kinds of inhibition Where – V 0 : velocity/rate of enzymatic activity – V max : The maximal rate of reaction when the enzyme is saturated – K m : (constant)the substrate concentration that produces ½ of the maximal velocity – S: substrate concentration

17. Lineweaver-Burk Plot Adaptation of Michaelis-Menten equation Yields a straight line

18. Influencing Factors on Enzymatic Reactions Substrate Concentration Enzyme Concentration – The higher the enzyme level, the faster the reaction pH – Most reaction occur in range of 7.0-8.0 – Changes in pH can denature an enzyme Temperature – Most reactions performed at 37 o C – Increasing temp increases rate of reaction – Avoid high/low temps due to denaturation of enzyme Cofactors – Influence the rate of reaction Inhibitors – Presence can interfere with a reaction can be reversible or irreversible

19. Types of Inhibition Competitive – Any substance that competes with the substrate for the active binding sites on the substrate – Reversible Non-competitive – Any substance that binds to an allosteric site Uncompetitive – Inhibitors bind to the ES complex – No product produced

20. Noncompetitive Inhibition Irreversible Inhibition Competitive Inhibition

21. Types of Inhibition Competitive Noncompetitive Uncompetitve

22. Enzyme Nomenclature Historical – ID of individual enzymes was made using the name of the substrate that the enzyme acted upon and adding “ase” as the suffix – Modifications were often made to clarify the reaction – International Union of Biochemistry (IUB) in 1955 appointed a commission to study and make recommendations on nomenclature for standardization

23. Enzyme Nomenclature: IUB Components – Systematic name Describes the nature of the reaction catalyzed Example: alpha 1,4-glucagon-4-gluconohydrolase – Recommended name Working or practical name Example: amylase – Numerical code First digit places enzyme in a class Second and third digit represent subclass(s) of the enzyme Fourth digit specific serial number in a subclass Example: 3.2.1.1

24. Enzyme Nomenclature: IUB Standard Abbreviated name – Accompanies recommended name – Example: AMS Common Abbreviated name – Example: AMY

26. Enzyme Classification: General Plasma vs. non-plasma specific enzymes – Plasma specific enzymes have a very definite/ specific function in the plasma Plasma is the normal site of action Concentration in plasma is greater than in most tissues Often liver synthesized Examples: plasmin, thrombin

27. Enzyme Classification: General – Non-plasma specific enzymes have no known physiological function in the plasma Some are secreted in the plasma Increased number of this type seen with cell disruption or death

28. Enzyme Classification Six classes – Oxidoreductases Involved in oxidation-reduction reactions Examples: LDH, G6PD – Transferases Transfer functional groups from one substrate to another Examples: AST, ALT – Hydrolases Catalyze the hydrolysis of various bonds Examples: acid phophatase, lipase

29. Enzyme Classification – Lyases Catalyze removal of groups from substrates without hydrolysis, product has double bonds Examples: aldolase, decarboxylase – Isomerases Involved in molecular rearrangements Examples: glucose phosphate isomerase – Ligases Catabolism reactions with cleavage of ATP Example: GSH

30. References Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins. http://regentsprep.org/Regents/biology/units/homeostasis/p rocesses.cfm http://student.ccbcmd.edu/~gkaiser/biotutorials/proteins/fg9.html http://student.ccbcmd.edu/~gkaiser/biotutorials/proteins/fg9.html Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson.