Transcript Lecture 15

Reaction Mechanisms
1.
The catalytically important amino acids are?
C, D, E, H, S WHY?
2.
In the protease mechanisms we have reviewed, the
carbonyl carbon on the peptide bond is the target.
•
3.
If you are given the catalytic amino acids of a protease,
remember the target and remember the products of the
protease reaction: 2 peptides
In the lysozyme mechanism, the reaction is started
by protonation of the glycosidic oxygen
•
We need to have products that have the hydroxyl groups
attached to them as we know carbohydrates should
(polyhydroxylaldehydes or ketones)
Chymotrypsin Mechanism
Different Active Site, Slightly Different
Mechanisms
Chymotrypsin is a protease, specifically a Serine Protease
There are other types of proteases:
1. Cysteine Proteases
•
Cys residue replaces Ser in mechanism similar to Serine
proteases
2. Aspartic Proteases
•
2 Asp residues act as General Acid-base catalysts
3. Zinc Proteases
•
Zn2+ is coordinated by 2 His
•
Zn2+ promotes attack of carbonyl carbon by water
Alcohol Dehydrogenase
Mechanism
Steps
• Binding of the coenzyme NAD+
• Binding of the alcohol substrate by coordination to
zinc
• Deprotonation of nicotinamide ribose by His-51
• Deprotonation of Ser-48 by nicotinamide ribose
• Deprotonation of the alcohol by Ser-48
• Hydride transfer from the alkoxide ion to NAD+,
leading to NADH and a zinc bound aldehyde or
ketone
• Release of the product aldehyde
Alcohol Dehydrogenase
Mechanism
Start at bottom and work your way
clockwise, Follow the electrons!
Alcohol Dehydrogenase
Questions for Your Consideration
1. How effective do you think the enzyme will
be with various alcohols as substrate?
2. What effect do you think performing the
reaction at an acidic pH would have? Basic
pH?
3. If you mutated Ser48 to a Threonine, what
would happen to the observed activity?
• Turn your answers in next Tuesday (March
9).
Membrane Function: Membrane
Transport
Passive transport
– driven by a concentration gradient
– simple diffusion: a molecule or ion moves
through an opening
– facilitated diffusion: a molecule or ion is carried
across a membrane by a carrier/channel protein
• Active transport
– a substance is moved AGAINST a concentration
gradient
– primary active transport: transport is linked to
the hydrolysis of ATP or other high-energy
molecule; for example, the Na+/K+ ion pump
– secondary active transport: driven by H+
gradient
Passive Transport
• Passive diffusion of species (uncharged) across
membrane dependent on concentration and the
presence of carrier protein
1˚ Active transport
• Movement of molecules against a gradient directly
linked to hydrolysis of high-energy yielding molecule
(e.g. ATP)
Membrane Receptors
• Membrane
receptors
– generally oligomeric
proteins
– binding of a
biologically active
substance to a
receptor initiates an
action within the cell
Oxidation Reactions
• Involves the transfer of electrons (OIL RIG):
– oxidation being termed for the removal of electrons
– reduction for gain of electrons
Loss of electrons or hydrogen = oxidation
Gain of electrons or hydrogen = reduction
• Oxidation is always accompanied by reduction of
an e- acceptor
• Cells (plants and animals) rely on O2 for life
processes
– Water an electron acceptor in plants
– Animal cells generate water from the reduction of O2 by
H+
Oxidation Reduction Reactions
Fe 2+ + Cu 2+  Fe 3+ + Cu +
Reaction can be expressed in the form of 2 half
reactions
Fe 2+  Fe 3+ + e- (oxidized); Fe 2+ = reducing agent
Cu 2+ + e-  Cu + (reduced) ; Cu 2+ = oxidizing agent
Reducing agent = e- donating molecule
Oxidizing agent = e- accepting molecule
They together make a conjugate redox pair.
Redox Potential
• Also known as oxidation reduction potential
• Redox potential of any substance is a measure of its
affinity for electrons
• In oxidation/reduction reactions the free energy change
is proportional to the tendency of reactants to donate /
accept e- denoted by E°’ ( for biological systems)
• A reaction with a positive E°’ has a negative Go’
(exergonic)
• The redox potential of a biological system is usually
compared with the potential of Hydrogen electrode
expressed at pH 7.0
Reduction potentials
•A reduction potential is a
measure of the affinity of an
atom for electrons
•Electrons are a standard
currency that let us rank the
reducing/oxidizing potential of
different redox couples.
•When the difference between
the E°’ values is positive, then
G° is negative because
G°=-nFE°’
•The more positive the
standard reduction potential
E°’, the greater the tendency
for the redox couple’s oxidized
form to accept electrons and
become reduced.
•Electrons flow towards the half
cell with the more positive
E°’
Reduction of NAD+ by FADH2
Consider the following reaction:
NAD+ + FADH2 --> FAD + NADH
+ H+
1st Half Reaction:
NAD+ + H+ + 2e- --> NADH
E°’ = -0.320V
2nd Half Reaction (Note: Its reversed!):
FADH2 --> FAD + 2H+ + 2eE°’ = +0.219V
E°’= –0.320V + +0.219V
= -0.101V.
Since E is negative, G is
positive and the reaction is not
spontaneous. Thus, FADH2
cannot be used to reduce NAD+.
Reduction of FAD by NADH
Consider the following reaction:
NADH + H+ + FAD --> FADH2 +
NAD+
1st Half Reaction (Note: Its reversed!):
NADH --> NAD+ + H+ + 2eE°’ = +0.320V
2nd Half Reaction:
FAD + 2H+ + 2e- --> FADH2
E°’ = -0.219V
E°’= +0.320V + -0.219V
= +0.101V.
Since E is positive, G is
negative and the reaction is
spontaneous. Thus, NAD+
can be used to reduce FADH2.