BCOR 011 Lecture 12

9/28/2005

ENZYMES
Last time…

-G reaction “can” go

spontaneous

But when will it go?

And at what rate?

Thermodynamics

Whether a reaction will occur

Kinetics

WHEN a reaction will occur

What governs WHEN a reaction will occur?

The tower of blocks falling is favorable

but when will it happen?

Oxidation of carbohydrate polymers (starch)

to carbon dioxide and water is favorable
but when will it happen?

Gasoline burning to carbon dioxide and water

is favorable
but when will it happen ?
For a Reaction to occur need to Destabilize Existing State
to INPUT ENERGY

Destabilization
energy input
“Activation
Energy”

Potential Potential
net net
usable usable
energy energy

Now In Transition
Need to INPUT ENERGY to Destabilize Existing State
Regain
Activation
Energy
Invested

Potential net
net usable
usable energy
energy released

In Transition After
 What does activation energy represent?
For a Reaction to Occur…

- reactants must find each other,

- meet in proper orientation
- and hit with sufficient force
 Productive
Collision

Many
Non-productive
Collisions
 Needs of Typical
chemical reactions
- need large number of molecular collisions

- need collide violently enough to

break pre-existing bonds (not bounce)

- need high concentration to find each

other at significant rate

HEAT !
 The energy profile for an exergonic reaction

A B

C D
Transition state

A B EA
Free energy

C D

Reactants
A B
∆G < O
C D

Products

Progress of the reaction

Figure 8.14
Temp 1 Temp 2 EA
Molecules with
sufficient
Energy (~40%)

Molecules with
sufficient
Energy (<5%)
ENZYMES make reactions
easier to occur at
reasonable temperature
by

LOWERING the
ACTIVATION ENERGY
EA
of the reaction
 Activation Energy
Energy necessary to overcome the status quo
“ease” of
EA initiating reaction

G Thermodynamic
“favorablility”

EA

G
 CATALYSTS:
promote a specific reaction
But are NOT consumed in the process

Key concepts:

Promotes - does not alter what would normally

occur thermodynamically
Specificity - promotes only one reaction, only
between specific reactants to give specific products

Reusable - regenerated in the process

ENZYMES are biological CATALYSTS

- usually PROTEINS

- sometimes RNA or
RNA/protein complexes
 Enzymes work as catalysts
by providing an easy path to the same point

Hard path

Easy path

HOW?
How do Enzymes do it?

1. Enzymes have BINDING AFFINITY

for their reactants = Substrates

Brings substrates in close proximity: conc

 Charged
Stabilized Interactions Nonpolar
Polar
Have a very Specific 3-D Shape

With a Specific Arrangement

of Functional Groups

Flexible
OH

HO
HO
+

Enzymes act as a Specific Platform

ENZYMES:

Bind ONLY specific things

HO
OH OH
Bind them ONLY in a OH HO
Specific 3-D Orientation HO -
+

SPECIFICITY is the Key to Enzyme Action

2. Enzymes ORIENT Substrates
always in productive orientation
 Productive
Collision

Many
Non-productive
Collisions

ONLY Productive
Collisions
With just a little nudge, can’t help but react

HO
OH OH
OH HO
HO -
+
3. Enzymes cause BOND STRAIN
- destabilize existing bonds
“nutcracker effect”

3a. Physical Strain

3b.Chemical Strain
The active site
– Is the region on the enzyme where the
substrate binds

Substate

Active site

Enzyme

Figure 8.16 (a)

Induced fit of a substrate

Enzyme- substrate
complex

Figure 8.16 (b)

Enzyme-substrate interactions

Fischer:
Lock & key

Koshland:
Induced fit

3a. Physical bond strain

Draw an quarter - an anvil
 • The catalytic cycle of an enzyme
1 Substrates enter active site; enzyme
changes shape so its active site 2 Substrates held in
embraces the substrates (induced fit). active site by weak
interactions, such as
hydrogen bonds and
ionic bonds.

3 Active site (and R groups of

Substrates Enzyme-substrate its amino acids) can lower EA
complex and speed up a reaction by
• acting as a template for
substrate orientation,
6 Active site • stressing the substrates
s available for and stabilizing the
wo new substrate transition state,
Mole. • providing a favorable
microenvironment,
Enzyme
• participating directly in the
catalytic reaction.

5 Products are
Released. 4 Substrates are
Converted into
Figure 8.17 Products Products.
3b. Chemical Bond Strain
tease the bond to fall apart
 Chemical Bond Strain

Stabilize a
Fictitious
state
 Cofactors
Non-polypeptide things at the active site
that help enzymes do their job

• Cofactors
– Are nonprotein enzyme helpers, eg Zn++

• Coenzymes
– Are organic cofactors
4. Enzymes “partake” in reactions
but are not consumed in them

Converts MANY “A’s” into “B’s”

 H+

OH-
H+

Partakes: but start and end with the same enzyme config
Lysozyme
Lysozyme: kills bacteria
Works at pH 4-5 Why?
 SUMMARY
Enzymes:
1. Bring reactants (substrates) in close proximity

2. Align substrates in proper orientation

3. Can act as a Lever: a press or an anvil

small shape change translates to large force

4. Release products when reaction done

rebind more substrates

5. Many small steps,

steps each easily achieved
rather than one huge leap
 Enzymes carry out reactions in a series of
small steps rather than one energetic event

No Problem
Dude
You expect
me to
JUMP this?
Reaction rates:

Example: H2O2-> H2O +O2

uncatalyzed –months

Fe +++ 30,000x faster

Catalase 100,000,000 x faster

 An enzyme catalyzed rxn
Can be “saturated”
Vmax
maximum velocity
Rate
or
velocity
1/2 Vmax

# made
per min

Km Substrate Conc
“substrate affinity”
The lower the Km the better
the enzyme recognizes substrate
“finds it at low conc”
“mpg”

The higher the Vmax the more

substrate an enzyme can
process per min
(if substrate around)
“top speed”
 Things that affect protein structure
often affect enzyme activity

temperature
0 20 40 60 80 100 ºC

pH

0 1 2 3 4 5 6 7 8 9 10
pH
Enzyme regulation:

Activity controlled

Continually adjusted
Principal Ways of Regulating Enzymes

Competitive Inhibition

Allosteric Inhibition

Covalent Modification (phosphorylation)

Competitive
HO
S1 S2
Inhibitors: OH
OH

-
bind to active site
I
HO
“unproductively” OH
and block
OH HO
true substrates’
access HO
+

S & I bind to same site

Competitive inhibition
Allosteric Inhibitors
“other” “site”

Distorts the conformation

of the enzyme

Negative
allosteric
regulator
Allosteric inhibition
Positive allosteric regulators

Helps enzyme work better

promotes/stabilizes an “active” conformation
Allosteric activation
Allosteric regulators change the shape
conformation of the enzyme
Allosteric activater
Allosteric enyzme Active site stabilizes active from
with four subunits (one of four)

Regulatory
site (one
of four) Activator
Active form Stabilized active form

Allosteric activater
stabilizes active form
Oscillation

Non- Inactive form Inhibitor Stabilized inactive

functional form
active
site

(a) Allosteric activators and inhibitors. In the cell, activators and inhibitors
Figure 8.20 dissociate when at low concentrations. The enzyme can then oscillate again.
A frequent regulatory modification
Phosphorylation of enzymes
 Phosphorylase kinase

inactive

+ P
active
Summary

1.enzymes are catalysts

2.Lower activation energy EA
3.Mechanism of action …
4.Enzyme kinetics- Vmax, Km
5.Regulation of enzyme activity
- competitive, allosteric
phosphorylation