Enzymes

Chapter 6

Enzymes Mostly Proteins (few RNA’s capable of catalysis)

Active Site - Substrate Binding + Reaction
S
Some require Cofactors
C f ((metals)) or Coenzymes
C ((organic cpds))

other binding sites are possible, see later regulation!

1
 Enzyme Pioneers Class Is First Part of E.C. Number

EC 2.7.1.1 = ATP:glucose phosphotransferase or Hexokinase

2 = Transferase
7 = Phosphotransferase
First Cell Free Prep First to Crystallize Urease Weak bonding at active 1 = Transferred to a hydroxyl
site results in catalysis
1 = Glucose is the acceptor

Enzyme Continuing with the EC Numbers-1

Search By
Class

Bacterial Luciferase
Rxn

FMNH2 + O2 + RCHO Î FMN + RCOOH + H2O + light

2
 Continuing with EC Numbers-2

NiceZyme

Enzyme with an Active Site Thermodynamics of a Reaction

S + E ÍÎ ES Î E + P

Active Site

Chymotrypsin

3
 Enzyme Catalyzed Reaction

E+S ↔ ES ↔ EP ↔ E+P

Dihydrofolate Reductase

NADP+

Folic Acid

4
 Substrate Binds in a Fold or Pocket Enzyme Reactions Bind Substrate then Change
Shape to Transition State

ΔGB = binding energy

Triose Phosphate Isomerase

Terribly Slow rate with Glyceraldehyde…phosphate

important in stabilizing binding.

5
Rate Enhancement Due to Proximity (Entropy
Reduction) Acid Catalysis

Acid Base Catalysis – Involve Proteins R groups Formation of a Covalent Intermediate

6
Michaelis Menten Curve

Michaelis Menten Equation:

L. Michaelis and Miss Maud L. Menten. 1913. "Die Kinetik der
Invertinwerkung" Biochemische Zeitschrift Vol. 49.

Vmax [S]
vo =
Km + [S]

Michaelis Menten Experiment

Determine Rate (v) at several concentrations of Substrate (S)
Here is one tube…one concentration of S

SÎP

Calculate Δ[S]/min .. this is for one [S]..this is catalytic rate

or Δ[P]/min.

7
Michaelis Menten Experiment: Real Data Textbook Figure – 5th Edition

Concentration of enzyme at
each [S] is exactly the SAME.

Calculate Δ[S]/min for each [S]

Do this at more concentrations

of S to get a larger data set
used for Î
next slide please

Michaelis Menten Curve

Michaelis Menten Equation is Non-Linear

Vmax [S]
vo =
Km + [S]

is Straightened Out by reciprocals…Lineweaver Burke

Equation:
1/vo = (Km/Vmax)(1/[S]) + 1/Vmax
the Equation of a Straight Line
y = mx + b

8
 Lineweaver-Burke Plot
There Are Other Equations to Convert Double Reciprocal
the Michaelis Menten Equation to a
Straight Line

Eadie Hofstie
v = -Km(v/S) + Vmax

Hanes Wolf:
S/v = (1/Vmax)(S) + Km/Vmax
Origin is Zero

all are y = mx + b

Vmax is an Extrinsic Property of Enzymes

At a high [S] :

Km = is an Intrinsic Property of an enzyme

What does this mean? Intrinsic vs Extrinsic?

9
 To get an Intrinsic Catalytic Constant from Vmax
kcat/Km

kcat comes from Vmax and [Enz] Vmax is [molar]/sec

[Enz] in molar
kcat = Vmax/ [Enz]

Calculation of Km and Vmax

Calculation of Km and Vmax
The enzyme, Practicase
Studentose Î Productate
Studentose Conc, mM 1/[S] Velocity, μmoles/ml/sec 1/v
Studentose, mM Velocity, μmoles/ml/sec
1 1 12 0.083
1 12
2 0.5 20 0.050
2 20
4 0.25 29 0.034
4 29
8 0.125 35 0.029
8 35
12 0.083 40 0.025
12 40
Assay volume = 1 ml
Now Plot this on Lineweaver Burk Plot….remember Zero is
near the middle of the graph!

10
 Lineweaver Burke Plot of Practicase Practicase kcat = an Intrinsic Property
1/
In the enzyme assay (one ml), each tube had 10 μg of
practicase. The molecular weight of practicase is 20,000
daltons.
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

1
or 10 μ sec.

Worked Example 6.1

An enzyme (happyase) is discovered to catalyze: So Far… … … it has just been:
SAD ÍÎ HAPPY
It is known that the kcat is 600 s-1. When [Et] = 20 nM,
and SAD = 40 μM, the reaction velocity (vo) is 9.6 μM/s.
S + E Î ES Î E + P
What is the KM?
Solution
S l ti combines
bi the
th kcat equation
ti and
d th
the Mi
Michaelis-
h li
Menten equation.
Now lets go to multiple substrates
kcat = Vmax/ [Et] so: Vmax = kcat [Et]
and products:
Substitute into Michaelis-Menten equation for Vmax:
vo = [ Vmax (S)] / [Km + (S)] A + B Î C + D
vo = [kcat [Et] (S)] / [Km + (S)] Answer: Km = 10μM

11
 Multiple Substrate Reactions Lineweaver Burke Plot – Enzymes forming
Ternary Complexes – Ordered or Random

Lineweaver Burke Plot – Enzymes Without

Ternary Complexes Reversible Inhibitors - Competitive

12
 Lineweaver Burke - Competitive Inhibition Reversible Inhibitors - Uncompetitive

α = 1 + [I]/Ki

-1/Km

-1/αKm

Lineweaver Burke – Uncompetitive Inhibition Reversible Inhibitors – Mixed Inhibition

α’ = 1 + [I]/Ki’

α’/Vmax

1/Vmax

These are not -1/Km, they are α’/Km

13
 Lineweaver Burke – Mixed Inhibition Shows Region of Inhibitor Effect

α = 1 + [I]/Ki
α’ = 1 + [I]/Ki’

α’/Vmax

Apparent Vmax or Apparent Km refers to y or x axis intercept only.

The Next Slide is MUCH BETTER
- 1/Km - α’/αKm

Calculation of Enzyme Constants Inhibition of Practicase

[Studentose],mM vo, uninhibited vo Inhbitor A vo Inhibitor B vo Inhibitor C

Type of Inhibition X axis intercept Y axis intercept
1 12 4.3 5.5 5
None -1/Km 1/Vmax 2 20 8 9 8.69
Competitive -1/αKm 1/Vmax 4 29 14 13 13.7

Uncompetitive -α
α’/K
/Km α’/Vmax
α /Vmax 8 35 21 16 19.6
12 40 26 18 22.2
Mixed - α’/αKm α’/Vmax

Inhibitor A at 1 mM
Inhibitor B at 3 mM
Inhibitor C at 50 μM

14
Practicase Inhbitiors
Calculation Inhibitor
1’s Practicase Ki :

Be sure to calculate
the Ki’s for the other
inhibitor.

Practicase Inhbitiors
Calculation of Mixed Inhibitor’s Ki’s
This is Inhibitor 2

This inhibitor has α and α’… to calculate Ki and Ki’

So, FIRST you need to calculate α’ … the best place to
do that is from the y-axis intercept = α’/Vmax
Then to get α, go to the x-axis intercept = α’/αKm
then from each, α and α’ you can determine Ki and Ki’

Remember this one inhibitor binds both to E and ES.

15
 Irreversible Inhibition
Enzymes and Fashion

“Stonewashed Jeans”

Jeans are washed with

cellulase (an enzyme that
hydrolyzes celluose –
major component in cotton) Mechanism Based
at a low concentration for a
short time…..the effect Suicide Inhibitors
looks “stonewashed”.
If they were really
stonewashed how would
they get all the stones out
of the pockets?

Effect of pH Chymotrypsin Effect of pH

16
 Chymotrypsin – Our Model Enzyme Chymotrypsin – Our Model Enzyme

Aromatic Part of
Substrate = Green

Chymotrypsin – Our Model Enzyme Chymotrypsin – Our Model Enzyme

Amide Nitrogens
Stabilize Oxyanion

Active Site

17
Reactive Groups in Enzymes are Either: Chymotrypsin – Our Model Enzyme

C terminal………N terminal

18
19
 Transition State Analogs!!!
Back to the Original Unmodified Enzyme May 10, 2004 C +E News

It works because it does not

stimulate other tissues such as
prostate or have other hyper-
stimulatory effects…that
steroids analogs do have.

Inhibits Plasminogen Activator

What about Antibiotic Resistance
Inhibitor-1 … to decrease
thrombosis (breaking down of
capillary walls)

Inhibits PDE-5 (like

Viagra, Levitra and
Cialis) which lowers
c-GMP levels Inhibitors of a transpeptidase that
builds bacterial cell wall
Inhibits Procollagen peptidoglycan-
Protease-C…reduces Enzyme call Penicillin Binding
scar tissue formation Protein

20
 Phenoxyacetal-Lactivicin + Penicillin Binding Protein
Drug Design
Company Ad

The importance of structural

protein chemistry !!!

from C&EN, Aug 13, 2007

Machebeouf et al. 2007. Structural and mechanistic basis of penicillin binding protein inhibition by lactivisions. Nature Chem Biol.
3:565-569

Hexokinase Reaction : Induced Fit Induced Fit with Glucose Binding

Daniel Koshland 60’s

What happens when glucose binds Î

21
 NMR Structure of Myoglobin
Enzyme Protein-Motion: Part of Catalysis

NMR is limited to
small proteins

From Chapter 4 Enzymes’ Many Movements. ACS Meeting News: C+E News April 20,2009 pg 34-36

Xylose is One Carbon Shorter than Glucose

Xylose causes Hexokinase to become an

ATPase

When Xylose reacts with Hexokinase – it causes induced fit

and Mg++ ATP binds…
but xylose does not exclude water from the active site
site.
Induced fit is the active form, and catalyses the phospho-
transfer from ATP to water Î ADP and Pi….futile use of ATP!

22
How Drugs Work – the Classic Penicillin Penicillin Inhibits Peptidoglycan Synthesis

Electron Microscopy Gm Pos and Gm Neg Envelopes Peptidoglycan Strands are Cross-linked

23
Penicillin Covalently Modifies Transpeptidase The Bacterial Response to Penicillin

Enzyme Regulation – Allosteric Effectors

The Human Response to the Bacterial Response

Does not inhibit

transpeptidase

24
 Aspartate Transcarbamoylase

12 Polypeptides
Active Sites

Allosteric Enzymes Often Have Sigmoid Kinetics

Feedback
Inhibition

25
Enzyme Regulation by Covalent Modification

26
Glycogen Synthase Regulation Zymogen Regulation

From Ch 15

27