Dr. Jeffrey P. Thompson bio350

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1 Chapter 8 Enzymes

3 Green light GFP Blue light

4 Modern day catalysis Catalysis (reaction promotion) may have gotten its beginning g in an RNA- dominated world. Most catalysis today has evolved into using PROTEIN enzymes to do the job. The capacity to specifically bind toavery wide range of molecules.

5 What do enzymes do Catalyze (accelerate) chemical reactions Maybe by a factor of more than a million Biological reactions require fast responses in the absence of enzymes, these reactions don t proceed at a perceivable rate. Carbonic anhydrase Hydrates 1 million CO2/sec 7,00, times faster than the uncatalyzed rate!

8 Protein break down is catalyzed by enzymes called PROTEASES.

9 Many proteases are also ESTERASES.

10 Trypsin specificity Thrombin specificity

13 What is so special about enzymes? The are specific. They enhance reactions. They minimize unwanted side reactions The specificity of an enzyme is due to the precise interaction of the substrate with the enzyme. This precision is a result of the intricate three-dimensional structure of the enzyme protein.

14 Many enzymes require Cofactors for activity Our discussion of the RNA-world mentioned that accessory molecules (peptides) may have enhance RNA s ability to do its many functions Today Some enzymes require accessory molecules to enhance their activity. cofactors Apoenzyme + cofactor = holoenzyme

15 Types of cofactors Metal ions Zn, Mg, Cu, Fe Small organic molecules Coenzymes (separate helpers ) vitamins i Prosthetic groups (physically attached helpers )

17 Enzyme classification Based on the TYPE OF REACTION THEY CATALYZE 1964 International Union of Biochemistry established an Enzyme Commission for categorization of enzymes. Like taxonomy for organisms A systematic way of identifying enzymes

18 Here are the major groups This is what they do

19 Thermodynamics and Enzymes Remember spontaneous reactions occur when G changes are negative. Spontaneous, products have less energy than reactants, EXERGONIC. Positive G changes are nonspontaneous, products have more energy than reactants, ENDERGONIC THE G OF A REACTION IS INDEPENDENT OF THE PATH OF THE TRANSFORMATION!

20 Thermodynamics and Enzymes The Standard free energy change of a reaction is related to the equilibrium constant G o Huh? In biology, reactions occur in different environments G OF A REACTION DEPENDS ON THE NATURE AND CONCENTRATIONS OF THE MOLECULES INVOLVED!!!!!

21 What do enzymes do? Important to note.. Enzymes alter only the REACTION RATE and not the Reaction equilibrium A B Forward rate constant t is 100 time that t of reverse reaction At equilibrium there is always a going to be 100 times more B than A Enzymes only help to reach equilibrium faster!

22 How do enzymes do this? Accelerate reactions by facilitating the formation of the transition state of the reaction. Enzymes decrease the Activation Energy, aka the free energy of activation. The essence of catalysis is SPECIFIC BINDING of the transition state inside the active site of the enzyme!

24 First step Formation of the Enzyme-substrate complex What is the evidence? SATURATION CURVES SUGGEST PHYSICAL CONTACT BETWEEN ENZYME AND SUBSTRATE. BIOPHYSICAL EVIDENCE FLUORESCENCE QUENCHING EXPERIMENTS NMR analysis

28 Common feature of Active Sites.. Catalytic groups (atoms from amino acids or cofactors in the active site that promote the reaction) Interaction of the enzyme and substrate at the active site promotes the FORMATION OF THE TRANSITION STATE!

29 Common features of Active Sites D cleft that is formed from groups that come from different parts of the amino acid sequence. 2. active sites take up a relatively small part of the total volume of the enzyme. 3. Active sites are clefts or crevices. 4. Substrates are bound to enzymes by multiple weak attractions. Why not covalent (strong) bonding? 5. Atoms in the active site give the specific binding.

30 Lysozyme active Site amino acids

32 Lock and Key

33 Induced fit

34 The observable kinetic properties can be described mathematically! Kinetic math models can explain the data Michaelis-Menten Model explains the data. Their model will describe the current model of enzyme reactions: E + S ES E + P Reaction velocity vs. Substrate conc. Vmax and Km values for enzymes

38 Km values reflect efficiency To get a reaction to proceed, substrate must bind Km values are the concentration of the substrate to give you half of the maximum velocity Smaller the Km value (concentration) the quicker the enzyme binds to the substrate Equals best efficiency (even binding efficiency of Enzyme to substrate)

40 Vmax values indicate speed Also called the Turnover number How many products are produced per second. Which enzyme on the next page has the greatest productivity?

41 AKA A.K.A K cat

42 Probing for information Testing the SAME ENZYME with DIFFERENT SUBSTRATES can give you some insight to the preferences of the active site. Calculate Km values for the different experiments and compare Look at the protease chymotrypsin for example.

44 Enzymatic Perfection

45 Many Biochemical Reactions Include Multiple Substrates 2 classes of multiple substrate reactions Sequential Displacement reactions Ordered sequential substrates bind in a defined sequence Random sequential No defined sequence. Double Displacement reactions Also called a ping pong reaction

46 Ordered SequentialDisplacement Rx LDH (h (the enzyme) Ternary Complex forms = Enzyme+ substrate 1+ substrate 2

47 Ordered Sequential Displacement Rx Cofactor adds first and leaves last Cleland Notation- method to represent An enzymatic reaction.

48 Random Sequential Displacement Rx Addition of substrates and leaving of products shows no order. Creatine kinase (the enzyme)

49 Sequential Random Displacement Rx Cleland Notation diagram is a little messier reflecting the different possibilities.

50 Double Displacement Rx AKA Ping-Pong reactions common for transferase enzymes Marked kdby a substituted enzyme complex (the enzyme is modified in the middle of the reactions) The two substrates take turns binding to active site-like watching A ping-pong match.

51 Double Displacement Rx Steps for this example: 1. enzyme binds aspartate (S) 2. enzyme removes amino group (holds onto it) 3. enzyme releases product (oxaloacetate)(p) 4. enzyme binds -ketoglutarate (S) 5. enzyme transfers amino group to -ketoglutarate 6. enzyme releases product glutamate(p) Marked by a substituted t enzyme complex

52 Allosteric Enzymes don t follow Michaelis-Menten Menten Kinetics Key Regulatory Enzymes

53 Enzyme Inhibitors Molecules that prevent the enzyme from working as normal They can cause changes in Vmax and Km Types Reversible inhibitors Noncovalently binds enzyme (bind and release) Irreversible inhibitors Covalently l bind enzyme (permanently attached)

54 Reversible inhibitors- Bound by noncovalent attachment Like the desired substrate both types of inhibitors can bind and release

55 These types of inhibitors Are Kinetically Discernable! They affect the kinetics curves Differently. The difference can be a Diagnostic tool to determine What kind of inhibitor is at work! Competitive=cause i Km values To increase, Vmax changes very Little! Noncompetitive=Km changes Very little but Vmax decreases Significantly!

56 A competitive Inhibitor it Resembles the Natural cofactor so it Can take its place! A cofactor For many enzymes

57 Lineweaver - Burk Plot

60 Irreversible inhibitors bind covalently to functional groups in enzymes. If the modified functional group is in an active site, the enzyme is permanently inactivated.

61 Another example.

62 Suicide Inhibition- inhibitor irreversibly binds to enzyme Only after being worked on by the enzyme! The enzyme is responsible for its own activation!

63 Remembering Transition States t

64 Transition state analogs are potent inhibitors of enzymes. 1. Remember what a transition state is? 2. Remember that enzymes catalyze reactions by promoting the formation of the transition state? t

65 Transition state analogs (molecules that resemble the transition State structure as the substrate is turning into product) are POTENT inhibitors. Do you think they would be competitive or noncompetitive? This is the hypothetical Transition state structure. This molecule, that resembles The transition state structure, Is a potent inhibitor of the enzyme used to turn L-proline Into its D form!

66 Generating a Man-made enzyme Proteins can be developed to catalyze reactions (like an enzyme) REMEMBER!! Enzyme function by promoting the formation of the transition state (this enhances the rate) If other proteins can be developed to bind to and stabilize a reaction s transition state, then they should serve as catalysts t as well! ABZYMES DO THIS!

67 ABZYMES - CATALYTIC ANTIBODIES How do antibodies work? Proteins in the immune system that naturally bind tightly to antigens to clear them from your body. Inject antigen in an animal = antibody production flu shot = anti-flu antibodies = protection against flu anti-flu antibodies bind to flu virus to immobilize it Inject a reaction s transition state analog (the antigen) develop antibodies that bind to the transition state These antibodies now CATALYZE THE REACTION!!

68 An Example Ferrochelatase enzyme needed d to add metal ions into Prosthetic groups needs to bend the group in the transition state. Use this antigen instead

Enzyme kinetics. Irreversible inhibition inhibitor is bound tightly to enzyme - very slow dissociation can be covalent or non covalently bound

Enzyme kinetics. Irreversible inhibition inhibitor is bound tightly to enzyme - very slow dissociation can be covalent or non covalently bound Enzymes can be regulated by acceleration and inhibition inhibition very common - several different mechanisms competitive / non competitive reversible / irreversible Irreversible inhibition inhibitor is