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Chemical reaction in cells are often coupled
Reaction 1- Glucose + Pi  Glucose-6-phosphate
(requires input of energy, endergonic)
Reaction 2- ATP  ADP + Pi
(releases energy, exergonic)
Reaction 1 + Reaction 2Glucose + ATP  Glucose-6-phosphate + ADP
(releases energy, exergonic)
Biochemical reactions are generally parts of metabolic pathways
Each step may not be energetically favorable, but the overall
pathway must be exergonic
Most small molecules are synthesized using such pathways
Enzymes are tightly regulated
Cells respond to their environment through the enzymes
Isoleucine starvation
Living organisms are broadly divided into two categories
Eukaryotes
(contain a nucleus)
and
Bacteria
(no nucleus)
~50 mm
Three domains of living organisms
Bacteria lack a nuclear membrane and are relatively simple
Cell envelope:
Contains cell wall
and membrane(s)
Gram positive:
one membrane
Eukaryotic cells are much more complex
A hydrogen bond is formed through electrical interaction
between an atom covalently bonded with a hydrogen and
which is more electronegative than hydrogen, and another atom
that has a partial negative charge.
Hydrogen bonds can also form between other functional groups
Despite their diversity, all proteins are made from 20 common
monomeric units called amino acids
O
C
Amino acids are carboxylic acids
OH
H
2
and contain an amino group
(Uncharged)
(Uncharged)
UV absorption
Phe
Cysteine can exist in two forms
Oxidation
Reduction
There is delocalization of electrons along the peptide bond
This creates,
1. An electric dipole moment within the peptide unit
2. Prevents rotation around the amide linkage
O
C
C
C
N
C
N
H
Trans
O
H
Cis
3. Makes each C-CO-NH-C flat
Proline does not have a strong preference for the trans configuration
4:1
The planar units around N-C and C-C (=O) bonds
Ramachandran Plot
Only certain combinations of f-y angles are “allowed”
Regular structures in proteins
-helix
=On•••H-Nn+4-
10
9
8
7
6
5
4
3
2
1
R-groups are on the outside
of the helix
Myohemerythrin
Helix bundle
b-sheet
b-strand
R-groups
above and
below
b-sheet
b-strand
R-groups
above and
below
Thioredoxin
Overall folding of the protein
The presence of disulfides reduces the number of alternate structures
Potential energy
Potential energy
Most enzymes are proteins
The exceptions are a handful RNA molecules that can act as enzymes
They are called ribozymes
Consequently, the rate of an enzymatic reaction is highest
when the enzyme is “saturated”
Rate
Maximum
velocity
[E] is constant
Enzymes are often aided by metals
Enzymes that use metals (other than Na+ and K+) are called
metalloenzymes
…...and by coenzymes
Energetics of enzyme action
E (enzyme)
S (substrate)  P (product)
[P]
Keq 
[S]
(Molar concentrations)
Keq is equillibrium constant
[P]
Keq 
[S]
Keq is equillibrium constant
G  -RT ln K
'0
'
eq
G is Gibbs free energy, G’0 is change in Gibbs free energy
under the following conditions: Temp 37°C (298 °K), pH 7.0,
1M reactants
R is Gas constant (8.3 J/mol or 2 Cal/mol)
All but the last condition is always satisfied
We may drop the superscript “’” and “0”, but it is always assumed
to be present
Michaelis-Menten kinetics
Also known as- Steady-state kinetics
k1
k2
E S ESE
P
k
-1
Constant
Assumptions: 1. There is a constant amount of enzyme-substrate
complex during the course of the reaction
Michaelis-Menten kinetics
Also known as- Steady-state kinetics
k1
k2
E S ESE
P
k
-1
Assumptions: 2. There is no reverse reaction
i.e. the reaction is irreversible
If you multiply both sides of the equation with k2 then...
k2 [ET ][S]
k 2 [ES] 
Km  [S]
k2[ES] = v (velocity of the reaction)
k2[ET] = k2[ETS] = vmax (the maximum velocity of the reaction)
v max[S]
v 
Km  [S]
Michaelis-Menten Equation
Turnover number (kcat) is defined as
kcat = vmax/[ET]
This defines the rate of catalysis per enzyme molecule
Specificity constant is defined as
kcat/Km
Types of enzyme inhibition
1. Competitive inhibition
Types of enzyme inhibition
2. Non-competitive inhibition
Types of enzyme inhibition
3. Irreversible inhibition
Other ways of regulating enzymes
1. Allosteric control
An enzyme is said to be regulated allosterically when
a molecule binds to a site other than the active site and modulates
enzyme activity
2. Covalent modifications
Enzymes are often (reversibly) covalently modified to change
there activity
3. Proteolytic cleavage
Some enzymes are activated by proteolytic cleavage
Schematic view of allosteric activation
Fraction of binding sites
occupied
Example of activation: Hemoglobin
Sigmoidal shape is
characteristic of
positive allosterism
Negative regulation of biochemical pathways
is often allosteric
Feedback Inhibition
Threonine dehydratase is negatively regulated by L-isoleucine
Covalent Modifications of Proteins
Perhaps the most important of these is phosphorylation
O-
Kinase
OH
+ ATP
O
P
O
O
-
+ ADP
Kinase
ATP
Active
ADP
Phosphatase
Inactive
Proteolytic cleavage of proteins
Purification of proteins
1. Size Exclusion chromatography
(aka Gel Filtration)
Separation of proteins by size
Larger (bigger) proteins are
“excluded” and go through faster
Smaller proteins are retained by
the beads and go through slower
Ion-exchange chromatography
In a cation exchanger, the
positively charged proteins
will tend to stick to the
column and move slowly
through the column.
In contrast, negatively
charged proteins will move
faster through the column
Opposite is true for the anion
exchangers.
2. Analysis of the subunit composition of the protein
A. Determination of MW by Gel filtration Chromatography
+
Proteins w ith know n MW
+
+
Relative
Elution
Volume
+
+
+
+
+
+
+
Log MW
Larger proteins elute first (in smaller elution volume)
B. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
O
Na+ -O
S
O
(CH2)11
CH3
O
Sodium dodecyl sulfate
HOH 2C
CH2 SH
2-mercaptoethanol
Breaks disulfide bridges
Polyacrylamide
matrix
1
2
3
4
5
6
Larger proteins elute later (migrate less distance on the gel)
3. Amino acid composition of the protein
Heat (>105°C)
24 Hr
Which amino acids are missing?
Tryptophan (May be accounted for by UV absorbance)
Gln
Asn
Glu
Asp
O
NH2
Acid Hydrolysis
O
O-
Hence,
Amt. of Glu (by acid hydrolysis) = Amt. of Glu + Amt. of Gln
Amt. of Asp (by acid hydrolysis) = Amt. of Asp + Amt. of Asn
4. Amino acid sequence of protein
Edman degradation