Ch 11!! Cont.

1. Ch 11!! Cont.

2. Classification of Enzyme • Commonly, enzymes were named by identifying the type of reaction and adding –ase. • As more enzymes were discovered, modifyers were placed before the name

3. Problems with Modifiers • Modifiers came from different things, such as: • Specific substrate • Enzyme source • Mode of regulation • Distinguishing feature of mechanism • Even then, more enzymes were discovered including multiple forms. • To handle this, alphanumeric designations were made such as Roman Numerals and greek alphabet

4. New System • To clear up the confusion, and address the increasing amount of new enzymes being discovered, the IUB system was created • The IUB system is very complex and some what mirrors species identification • It gives each enzyme a unique name and a code number that reflects the type of reaction catalyzed and the substrate involved.

5. IUB naming system • As a result, enzymes are now grouped into 6 classes by reaction type and each class has multiple subclasses, etc • Example: • ATP: D-Hexose-6-phosphatetransferase E.C.2.7.1.1 • 2= transferase, 7=transfer of phophoral group, 1=alcohol in phosphoral acceptor

6. IUB cont • Parts of the name provide information as well • In the last case, the hexose-6 tells us the alcohol phosphorylated is on carbon 6 of hexose

7. Six major classes • 1- Oxidoreductases • 2- Transferases • 3- Hydrolases • 4-Lyases • 5- Isomerases • 6- Ligases • Examples on page 393.

8. Non Protein Material • Enzymes often contain non-protein materials as well • These are termed prosthetic groups, cofactors, or coenzymes • All basically expand the enzymes capabilities beyond the traditional functionality found in proteins

9. Definitions • Prosthetic groups- groups that are tightly bound to the enzyme either by covalent or non-covalent interactions • Metal are the most common • About 1/3 of all enzymes are termed metalloenzymes because they contain metals

10. Definitions • Cofactors- differ from prosthetic groups in that they are not permanently attached. • They bind to either the substrate or enzyme to perform the reaction the move on • Again, metals are most common • Difference is they are not bound!!! • The term metal-activated enzymes refers to enzymes that need metals as cofactors.

11. Definitions • Coenzymes- transporters. They don’t catalyze a reaction, just move things from where they were made to where they are used. • Coenzymes can protect and stabilize substrates until they are needed. • The B vitamins supply components for numerous coenzymes, Table 11.5 page 389

12. The Active Site • The Active Site in enzymes is the specific location of action. It is usually a small portion of the enzyme. • The Active site: • Gives high selectivity • Usually forms a cleft or pocket • Involves groups from multi-monomer units • Binds to non-reacting portion of substrate • Also orientates/binds cofactors/prosthetic groups that are needed

13. Enzymatic Mechanisms • There are 4 general mechanisms that account for an enzymes ability to catalyze reactions • Catalysis by Proximity -molecules have to come together to react! - Typically done by increasing the local concentration - Binding increases the local concentration

14. Enzymatic Mechanisms 2) Acid/Base Catalysis - The amino acid side groups and/or prosthetic groups can act as acids or bases - There are two types: a) specific acid/base catalysis b) general acid/base catalysis

15. Enzymatic Mechanisms 3) Catalysis by Strain -Usually the mechanism for breaking bonds -typically bends or orientates bonds into unfavorable conformations 4) Covalent Catalysis -a covalent bond is created between the enzyme and substrate -usually present in group transfers -the covalent bond is temporary

16. Conformational Change in Enzyme • Early on, the lock and key analogy was used to explain enzyme activity • But this inferred rigidity in the enzyme that experimental evidence didn’t support • Later, the hand and glove analogy was used • When the substrate binds, enzymes change slightly which allows for catalytic acitivity • Figure 11.7 and 11.8, page 369

17. Examples of Acid/Base Catalysis • Board

18. Example of Covalent Catalysis • Board and Figure 11.13, page 374

19. Catalytic Residues are highly conserved • Most enzymes in particular family’s use the same mechanism on different substrates • It is believed that genes are duplicated to create the individual proteins • Use of more than one gene to encode proteins allows for each to form independently which accounts for different binding properties

20. When different proteins have similar residues in the same position, the residues are said to be conserved residues • Proteins with a large number of conserved residues are said to be homologous

21. Isozymes • Isozymes are different enzymes that catalyze the same reaction • The difference may be in certain properties that adapt them to specific tissues or locations • Other differences may be higher selectivity • Ex. Hexokinasevsglucokinase • Could also just supply back up copies of essential enzymes

22. Detection of enzymes • Enzymes are often present in very small amounts • This make detection and quantification difficult • Instead of trying to isolate and identify the actual enzyme, often we just look for evidence of their presence • Amount of enzyme present can sometimes be determined by the rate of the catalyzed reaction.

23. Detection of proteins that lack catalytic activity is a little more complicated • For these cases, Enzyme-Linked ImmunoAssays (ELISA’s) are used • There are two ways to do ELISA’s with the difference being what is bound.

24. ELISA’s • Step 1- adhere the protein • Step 2- add an antibody with a reporter enzyme • Step 3- antibody binds to immobilized protein • Step 4- use the catalytic activity of the reporter enzyme to determine the presence and quantity of the original protein

25. Other methods are also available depending on the properties of the reactants and products of the reaction being catalyzed. • This method is used to assay NAD(P)+ dependant dehydrogenases. • Example:

26. Other Alternatives • If your reactant/product is not accompanied by a change in absorption or fluorescence, the assay is generally more difficult • Often, you are required to separate the products from the substrates prior to measuring • Another option is to create a synthetic substrate whose product can be detected

27. Lastly, in some instances, the product of the reaction of interest can be transformed into something that is readily detectible by coupling processes.

28. Why do we quantify/detect enzymes • There are thousands of different enzymes • Some of them function all the time and are essential in the vitality of the cell and are present throughout the body • Other enzymes or isozymes are used only in specific cells, or during specific times of development, or in response to specific physiologic or pathologic changes

29. Analysis of these enzymes often aids in diagnosis. • Example: Non-functional plasma enzymes • In addition, different forms of the same enzymes can differentiate between multiple problems • Example- Isozymes of Lactate Dehydrogenase

30. Enzyme Kinetics • Enzyme Kinetics- the quantitative measurement of the rates of enzyme catalyzed reactions and the systematic study of factors that affect rates • By studying the kinetics of a process we can deduce the mechanism • Knowing the mechanism helps us find ways to either promote or prevent the process.

31. Temperature • Raising the temperature increases the kinetic energy of molecules. • The total number of molecules whose kinetic energy exceeds the energy barrier for the formation of products increases as we go from low temp to medium temp to high temp. • Increasing the KE of molecules also increases their motion and therefore the frequency with which they collide • This combination of more frequent and more highly energetic and productive collisions increases the reaction rate.

32. Reactant Concentration • The frequency with which molecules collide is directly proportionate to their concentration • At constant temperature, the number of molecules with enough energy to overcome the energy barrier is constant but the rate is proportionate to the number of collisions, therefore, to the molar concentration.

33. Reactant Concentration • The coefficients therefore becomes a power in the rate equation. • In general: nA + mB → P Rate ∞ [A]n[B]m By including a rate constant, the proportion sign is replaced by an equal sign Rate = k[A]n[B]m

34. Rate Equations • Remember, most reactions are reversible, so the reverse equation would be: Rate = k-1[P] • At equilibrium, the total concentration of reactants and products is constant • Another way to state this is that the rate of the forward reaction is equal to the rate of the reverse reaction.

35. Equilibrium Constant • The ratio of k1 to k-1 is termed the equilibrium constant with the symbol keq • There are four important properties of systems at equilibrium which must be considered: • keq is a ratio of rate constants, not rates! • The reaction rates are equal, not the rate constants

36. 3) Equilibrium is a dynamic state. There is no net change in concentration, but continual interconversion 4) The numeric value for keq can be calculated from either the ratio of k1 to k-1 or from the concentration of reactants and products

37. How enzymes work • We have said before that enzymes speed up reactions by lowering the activation energy • They do this by lowering the energy of the transition state • When the mechanism is the same as the uncatalyzed reaction, the environment of the active site lowers ∆GF by stabilizing the intermediates

38. Stabilization • There are 3 ways for this to occur: • acid/base groups assist in transfer of H+ to or from intermediates • Positioned charged groups or metal ions stabilize developing charges • Creating or relieving steric strain

39. Stabilization • If the reaction proceeds via a different mechanism than the typical reaction, it is usually covalent catalysis!

40. Enzymes and keq • Enzymes DO NOT effect keq • Enzymes increase rates only be lowering activation energies • They do no effect ∆G, keq, etc

41. Measuring Enzyme Catalyzed Reaction Rates • These are measured over very short periods of time • There is a huge excess of substrate compared to enzyme • Under these conditions, only small amounts of product are produced • But by doing this, the reverse reaction is negligible, so the initial velocity (vi) is essentially that of the desired forward reaction

42. One important factor is that vi is proportionate to the enzyme concentration • This provides two things: • In lab, we can increase or decrease the reaction rate by altering the enzyme concentration • By determining a concentration that gives a rate comparable to in the cell, we can determine enzyme concentration in the cell

43. Substrate Concentration • Substrate concentration can also affect reaction rates • As you increase the substrate concentration, vi increases until it reaches a maximum value, vmax • Once vmax is reached, increasing substrate concentration has no affect • The reasoning is the relationship between the substrate and enzymes

44. Vmax • The substrate must bind to the enzyme to form the ES complex • Only substrate in the ES complex can be converted to product • The equilibrium constant for forming the ES complex is not infinitely large so even if the substrate is in excess, only a fraction will form the ES complex

45. Vmax • So as you increase the substrate concentration, you are also increasing that fraction forming the ES complex • However, once Vmaxis reached, all enzymes are in ES complexes and increasing concentration of substrate will not increase the rate because no more enzyme is available for binding. • At this point, vi depends solely on the ability of free enzyme to be released.

46. Inhibitors • Inhibitors are classified by: • The site of action on the enzyme • Whether or not they chemically modify the enzyme • Kinetic parameters they influence • There are two major Classes: Reversible Inhibition and Irreversible Inhibition

47. Reversible Inhibition • Kinetically, there are two classes based on whether raising the substrate concentration does or does not overcome inhibition • Competitive Inhibitors- typically resemble the substrate, bond to the active site, and acts by decreasing the number of free enzymes available to bind substrates • A double reciprocal plot helps evaluate inhibitors.

48. Kinetically • Noncompetitive Inhibitors- binding of the inhibitor does not effect binding of the substrate. • The inhibitor binds to a second site (not the active site) • By binding at the second site, it somehow lowers the efficiency in which the enzyme converts the substrate to product.

49. Regulation of Enzyme Activity • Homeostasis is very important • This is our ability to adapt to both changes in the internal and external environment • We respond to these changes by balanced, coordinated changes in the rates of specific metabolic reactions • Many diseases are characterized by creating certain dysfunctions in this regulatory procedure

50. If enzymes operated at their saturated levels, they would not be able to adjust to more substrate • For this reason, most substrate concentrations are maintained near the Km value.