Slide 1

Chapter 6 Protein Function : Enzymes Part 1

Slide 2

Enzymes Physiological significance of enzymes Catalytic power of enzymes Chemical mechanisms of catalysis Mechanism of chymotrypsin Description of enzyme kinetics and inhibition Enzyme Learning Goals: to Know

Slide 3

Enzymes Mostly Proteins (a few RNAs are capable of catalysis) Active Site: Substrate Binding + Reaction Products Some require Cofactors (metals) or Coenzymes (organic cpds) Some enzymes have other binding sitesinvolved in regulation, we will see later, Part 2 EOC Problem 1 involves the sweetness of corn affected by corn enzymes and Problem 2 calculates the average molar concentration of enzymes in a bacterial cell: you can take it further to find the number of molecules of each enzyme present in a cell.

Slide 4

Slide 5

Slide 6

First Cell Free Prep First to Crystallize Urease Weak bonding at active site results in catalysis Enzyme Pioneers

Slide 7

Why biocatalysis over inorganic catalysts? Greater reaction specificity: avoids side products Milder reaction conditions: conducive to conditions in cells Higher reaction rates: in a biologically useful timeframe Capacity for regulation: control of biological pathways Metabolites have many potential pathways of decomposition Enzymes make the desired one most favorable EOC Problem 4: Examines the thermal protection of hexokinase that a substrate brings to the table: maintaining conformation under harsh conditions. Later in Part 2 of this chapter we will see X ray data backing this up.

Slide 8

Enzymatic Substrate Selectivity: Phenylalanine hydroxylase No binding Binding but no reaction

Slide 9

Class Is the First Part of E.C. Number EC 2.7.1.1 = ATP:glucose phosphotransferase or Hexokinase 2 = Transferase 7 = Phosphotransferase 1 = Transferred to a hydroxyl 1 = Glucose is the acceptor

Slide 10

Enzyme Search By Class FMNH 2 + O 2 + RCHO FMN + RCOOH + H 2 O + light Bacterial Luciferase Rxn

Slide 11

Continuing with the EC Numbers-1

Slide 12

Continuing with EC Numbers-2

Slide 13

NiceZyme

Slide 14

Enzyme with an Active Site Chymotrypsin Active Site

Slide 15

Thermodynamics of a Reaction S + E ES E + P

Slide 16

Enzyme Catalyzed Reaction E + S ES EP E + P

Slide 17

Slide 18

EOC Problem 3: A rate enhancement problem using Urease, the enzyme that converts: Urea CO 2 + 2 NH 3. The calculation demonstrates how long it would take if urease were not present !

Slide 19

Dihydrofolate Reductase Substrate Binds in a Fold or Pocket G B = binding energy Folic Acid NADP + +

Slide 20

Enzyme Reactions Bind Substrate then Change Shape to Transition State

Slide 21

Slide 22

Triose Phosphate Isomerase Terribly Slow rate with Glyceraldehydephosphate important in stabilizing binding.

Slide 23

Rate Enhancement Due to Proximity (Entropy Reduction)

Slide 24

Acid/Base Catalysis

Slide 25

Catalytic Mechanisms acid-base catalysis: give and take protons covalent catalysis: change reaction paths metal ion catalysis: use redox cofactors, pK a shifters electrostatic catalysis: preferential interactions with Transition State.

Slide 26

Acid Base Catalysis Involve Proteins R groups

Slide 27

Formation of a Covalent Intermediate

Slide 28

Michaelis Menten Curve

Slide 29

Michaelis Menten Equation: V max [S] K m + [S] v o = L. Michaelis and Miss Maud L. Menten. 1913. "Die Kinetik der Invertinwerkung" Biochemische Zeitschrift Vol. 49. Invertase Reaction: sucrose + H 2 O glucose + fructose

Slide 30

Slide 31

Michaelis Menten Experiment Measure Rate (v) at several concentrations of Substrate (S) Here is one tube with one beginning concentration of S Calculate [S]/min or [P]/min. S P E This enzyme, triosephosphate isomerase is a one substrate, one product enzyme.

Slide 32

Michaelis Menten Experiment: Real Data At each [S], the concentration of enzyme is exactly the SAME. Calculate [S]/min for each [S] EOC Problem 6 is about using 340nm light to measure dehydrogenase reactionsthe classic lactate dehydrogenase. Do this at more concentrations of S to get a larger data set used for

Slide 33

Initial Velocites are the Dashed Line A

Slide 34

Michaelis Menten Plot

Slide 35

Michaelis Menten Equation is Non-Linear Straightened Out by reciprocalsLineweaver Burke Equation: 1/v o = (K M /V max )(1/[S]) + 1/V max the Equation of a Straight Line y = mx + b Thus, y = 1/v o, x = 1/[S] and m (the slope) = K M /V max Lets Plot this Outnext slide V max [S] K M + [S] v o =

Slide 36

Lineweaver-Burke Plot Double Reciprocal Origin is Zero Data Points are in this range

Slide 37

There Are Other Equations to Convert the Michaelis Menten Equation to a Straight Line Eadie Hofstie v = -Km(v/S) + V max Hanes Wolf: S/v = (1/V max )(S) + Km/V max all are y = mx + b

Slide 38

K M = is an Intrinsic Property of an enzyme What does this mean? Intrinsic vs Extrinsic?

Slide 39

V max is an Extrinsic Property of Enzymes At a high [S], varying only the enzyme conc :

Slide 40

k cat comes from V max and [Enz] V max is [molar]/sec [Enz] in molar To get an Intrinsic Catalytic Constant from V max k cat = V max / [Enz]

Slide 41

k cat /K m

Slide 42

Calculation of Km and Vmax The enzyme, Practicase Studentose Productate Studentose, mM velocity, moles/ml/sec 112 220 429 835 1240 Assay volume = 1 ml/tube Whats in the tube: buffer + enzyme, then add substrate at time Zero. EOC Problems 11(dead easy to do by inspection) and 13 to do by Lineweaver Burke plot

Slide 43

Calculation of Km and Vmax Studentose, mM 1/[S] Velocity, 1/v moles/ml/sec 1 112 0.083 2 0.520 0.050 4 0.2529 0.034 8 0.12535 0.029 12 0.08340 0.025 Now Plot this on Lineweaver Burk Plot.remember Zero is near the middle of the graph!

Slide 44

Lineweaver Burke Plot of Practicase 1 1/

Slide 45

Practicase k cat = an Intrinsic Property In the enzyme assay (one ml), each tube had 10 g of practicase. The molecular weight of practicase is 20,000 D. Thus, each tube had 10 g / 20,000 g/mole = 0.0005 mole practicase k cat = V max / [Enz] = (50 mole/sec)/ 0.0005 mole = 1 x 10 5 s -1 Thus one enzyme reaction takes 1/ 1x 10 5 s -1 = 10 -5 sec or 10 sec.

Slide 46

What is Wrong with this L-B graph?

Slide 47

Slide 48

Things to Know and Do Before Class 1.Principles of catalysis. 2.Enzyme naming concepts. 3.Intrinsic and Extrinsic values of Enzyme kinetics. 4.Be able to do a Michaelis Menten graph. 5.Be able to do a Lineweaver Burke graph. 6.To do EOC problems 1-6, 11, 13.