Chapter 6 Protein Function : Enzymes Part 1

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Chapter 6Protein Function Enzymes Part 1
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Enzymes
Enzyme Learning Goals to Know

• Physiological significance of enzymes
• Catalytic power of enzymes
• Chemical mechanisms of catalysis
• Mechanism of chymotrypsin
• Description of enzyme kinetics and inhibition

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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.
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Enzyme Pioneers
First Cell Free Prep First to Crystallize
Urease Weak bonding at active
site results
in catalysis
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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.
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Enzymatic Substrate Selectivity Phenylalanine
hydroxylase

• No binding

Binding but no reaction
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Class Is the First Part of E.C. Number
EC 2.7.1.1 ATPglucose phosphotransferase or
Hexokinase 2 Transferase 7
Phosphotransferase 1 Transferred to a
hydroxyl 1 Glucose is the acceptor
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Enzyme Search By Class
Bacterial Luciferase Rxn
FMNH2 O2 RCHO ? FMN RCOOH H2O light
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Continuing with the EC Numbers-1
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Continuing with EC Numbers-2
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NiceZyme
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Enzyme with an Active Site
Active Site
Chymotrypsin
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Thermodynamics of a Reaction
S E ?? ES ? E P
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Enzyme Catalyzed Reaction
E S ? ES ? EP ? E P
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EOC Problem 3 A rate enhancement problem using
Urease, the enzyme that converts Urea ? CO2
2 NH3. The calculation demonstrates how long
it would take if urease were not present !
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Dihydrofolate ReductaseSubstrate Binds in a Fold
or Pocket
NADP

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Folic Acid
?GB binding energy
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Enzyme Reactions Bind Substrate then Change Shape
to Transition State
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Triose Phosphate Isomerase
Terribly Slow rate with Glyceraldehydephosphate
important in stabilizing binding.
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Rate Enhancement Due to Proximity (Entropy
Reduction)
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Acid/Base Catalysis
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Catalytic Mechanisms

• acid-base catalysis give and take protons
• covalent catalysis change reaction paths
• metal ion catalysis use redox cofactors, pKa
  shifters
• electrostatic catalysis preferential
  interactions with Transition State.

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Acid Base Catalysis Involve Proteins R groups
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Formation of a Covalent Intermediate
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Michaelis Menten Curve
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Michaelis Menten Equation
L. Michaelis and Miss Maud L. Menten. 1913. "Die
Kinetik der Invertinwerkung" Biochemische
Zeitschrift Vol. 49.
Vmax S Km S
vo
Invertase Reaction sucrose H2O ? glucose
fructose
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Michaelis Menten Experiment
Measure Rate (v) at several concentrations of
Substrate (S) Here is one tube with one beginning
concentration of S
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S ? P
This enzyme, triosephosphate isomerase is a one
substrate, one product enzyme.
Calculate ?S/min or ?P/min.
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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 ?
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Initial Velocites are the Dashed Line
A
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Michaelis Menten Plot
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Michaelis Menten Equation is Non-Linear
Vmax S KM S
vo
Straightened Out by reciprocalsLineweaver Burke
Equation 1/vo (KM/Vmax)(1/S) 1/Vmax the
Equation of a Straight Line y mx b Thus, y
1/vo , x 1/S and m (the slope)
KM/Vmax Lets Plot this Outnext slide
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Lineweaver-Burke PlotDouble Reciprocal
Data Points are in this range
Origin is Zero
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There Are Other Equations to Convertthe
Michaelis Menten Equation to a Straight Line
Eadie Hofstie v -Km(v/S) Vmax Hanes
Wolf S/v (1/Vmax)(S) Km/Vmax all are y
mx b
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KM is an Intrinsic Property of an enzyme What
does this mean? Intrinsic vs Extrinsic?
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Vmax is an Extrinsic Property of Enzymes
At a high S, varying only the enzyme conc
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To get an Intrinsic Catalytic Constant from Vmax
kcat comes from Vmax and Enz Vmax
is molar/sec Enz in molar
kcat Vmax/ Enz
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kcat/Km
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Calculation of Km and Vmax
The enzyme, Practicase Studentose ?
Productate Studentose, mM velocity,
µmoles/ml/sec 1 12 2 20 4 29 8
35 12 40 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
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Calculation of Km and Vmax
Studentose, mM 1/S
Velocity, 1/v
µmoles/ml/sec 1 1 12
0.083 2 0.5 20 0.050 4
0.25 29 0.034 8 0.125 35
0.029 12 0.083 40
0.025 Now Plot this on Lineweaver Burk
Plot.remember Zero is near the middle of the
graph!
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Lineweaver Burke Plot of Practicase
1/
1
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Practicase kcat an Intrinsic Property
In the enzyme assay (one ml), each tube had 10 µg
of practicase. The molecular weight of
practicase is 20,000 D. Thus, each tube had 10
µg / 20,000 µg/µmole 0.0005 µmole
practicase kcat Vmax/ Enz (50 µmole/sec)/
0.0005 µmole 1 x 105 s-1 Thus one enzyme
reaction takes 1/ 1x 105 s-1 10-5 sec or
10 µ sec.
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What is Wrong with this L-B graph?
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What is Wrong with this L-B graph?
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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.