chapter 6 protein function part 2: enzymes
TRANSCRIPT
Chapter 6
Protein Function : Enzymes
Part 1
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
Enzymes
Mostly Proteins (a few RNA’s are capable of catalysis)Active Site: Substrate Binding + Reaction Products
Some require Cofactors (metals) or Coenzymes (organic cpds)
Some enzymes have other binding sites…involved 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.
First Cell Free Prep First to Crystallize Urease Weak bonding at active site results in catalysis
Enzyme Pioneers
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
COO
OH
O COO
COO
O COO
NH2
OOCCOO
O
OH
OH
COO
NH2
COO
-
-
-
-
-
-
--
Chorismate mutase
• 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.
Enzymatic Substrate Selectivity: Phenylalanine hydroxylase
No binding
OOC NH3
H
OOC NH3
H
H NH
HOH
OH
H
OH
CH3
OOC NH3
HOH
--
-
+
+
+
Binding but no reaction
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
Enzyme Search By
Class
FMNH2 + O2 + RCHO FMN + RCOOH + H2O + light
Bacterial Luciferase Rxn
Continuing with the EC Numbers-1
Continuing with EC Numbers-2
NiceZyme
Enzyme with an Active Site
Chymotrypsin
Active Site
Thermodynamics of a Reaction
S + E ES E + P
Enzyme Catalyzed Reaction
E + S ↔ ES ↔ EP ↔ E + P
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 !
Dihydrofolate ReductaseSubstrate Binds in a Fold or Pocket
ΔGB = binding energy
Folic Acid
NADP+
+
Enzyme Reactions Bind Substrate then Change Shape to Transition State
Triose Phosphate Isomerase
Terribly Slow rate with Glyceraldehyde…phosphate important in stabilizing binding.
Rate Enhancement Due to Proximity (Entropy Reduction)
Acid/Base Catalysis
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.
Acid Base Catalysis – Involve Proteins R groups
Formation of a Covalent Intermediate
Michaelis Menten Curve
Michaelis Menten Equation:
Vmax [S]
Km + [S]vo =
L. Michaelis and Miss Maud L. Menten. 1913. "Die Kinetik der Invertinwerkung" Biochemische Zeitschrift Vol. 49.
Invertase Reaction: sucrose + H2O glucose + fructose
Michaelis Menten ExperimentMeasure Rate (v) at several concentrations of Substrate (S)
Here is one tube with one beginning concentration of S
Calculate Δ[S]/min or Δ[P]/min.
S PE
This enzyme, triosephosphate isomerase is a one substrate, one product enzyme.
Michaelis Menten Experiment: Real Data
At each [S], the concentration of enzyme is exactly the SAME.
Calculate Δ[S]/min for each [S]
EOC Problem 6 is about using 340nm light to measure dehydrogenase reactions…the classic lactate dehydrogenase.
Do this at more concentrations of S to get a larger data set used for
Initial Velocites are the Dashed Line
A
Michaelis Menten Plot
Michaelis Menten Equation is Non-Linear
Straightened Out by reciprocals…Lineweaver Burke Equation:
1/vo = (KM/Vmax)(1/[S]) + 1/Vmax
the Equation of a Straight Liney = mx + b
Thus, y = 1/vo , x = 1/[S] and m (the slope) = KM/Vmax
Lets Plot this Out…next slide
Vmax [S]
KM + [S]vo =
Lineweaver-Burke PlotDouble Reciprocal
Origin is Zero
Data Points are in this range
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
KM = is an Intrinsic Property of an enzyme
What does this mean? Intrinsic vs Extrinsic?
Vmax is an Extrinsic Property of Enzymes
At a high [S], varying only the enzyme conc :
kcat comes from Vmax and [Enz] Vmax is [molar]/sec
[Enz] in molar
To get an Intrinsic Catalytic Constant from Vmax
kcat = Vmax/ [Enz]
kcat/Km
Calculation of Km and VmaxThe enzyme, Practicase
Studentose Productate
Studentose, mM velocity, μmoles/ml/sec
1 12
2 20
4 29
8 35
12 40Assay volume = 1 ml/tube
What’s 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
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!
Lineweaver Burke Plot of Practicase
1
1/
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.
What is Wrong with this L-B graph?
What is Wrong with this L-B graph?
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.