Metabolic Biochemistry

BIBC 102
Fall 2013
Lecture 3

October 2, 2013
Protein Synthesis

Apoenzyme

+ Active enzyme

Co-enzyme
Co-factor
Prosthetic group
Metal ion(s)
Ca2+ structural and regulatory role in many enzymes
 NOMENCLATURE

EC3.4.17.1: hydrolase -
peptidase (subclass) -
metallocarboxypeptidase (sub-subclass) -
arbitrary assigned serial number
METABOLIC CHART
SIGMA
 KEGG

Kyoto Encyclopedia of Genes and Genomes

http://www.callutheran.edu/Academic_Programs/Departments/BioDev/omm/gallery.htm
 Regulation of enzyme activity
1. At the level of gene expression and protein turnover

2. By covalent modifications of side chains

3. By non-covalent binding of small molecules (ligands)

that are not substrates

4. Allosteric mechanisms in multisubunit enzymes

The mechanisms 2-4 are elucidated by the investigation

of the kinetics of enzymatic reactions
1. At the level of gene expression and protein turnover

DNA (Gene)

transcription

mRNA
translation

Protein
turnover (degradation)
2. By covalent modifications of side chains

3. By non-covalent binding of small molecules (ligands) that

are not substrates

4. Allosteric mechanisms in multisubunit enzymes

Stay Tuned
 ENZYME KINETICS

ENZYME ASSAYS

LNC Chapter 6
REACTANTS
PRODUCTS
SUBSTRATES

k
First order reactions: A  P

k
Second order reactions: A+B  P
 k
First order reaction: A  P

d[P]/dt = - d[A]/dt = k[A]

ln[A] = ln[Ao] - kt or [A] = [Ao] exp(-kt)

 a reversible first order reaction:

k1
A P
k-1

d[P]/dt = k1 [A] - k-1 [P] = 0 at equilibrium

k1 [A] = k-1 [P]

k1/k-1 = Keq = [P] /[A]

Go = - RT ln Keq = - RT ln [P]/[A]

 k
Second order reaction: A+B  P

d[P]/dt = -d[A]/dt = -d[B]/dt = k[A][B]

When [A] = [B] or when A + A  products

d[P]/dt = -d[A]/dt = k[A]2

1/[A] = 1/[Ao] + kt
 [A]o
When plotted as [A] vs. time, first and second
order plots look very similar to the untrained eye.

[A]

time
ln[A]o
slope = k
slope = - k 1/[A]
ln[A]

2nd order
1st order 1/[A]o

time time
 TRANSITION STATE THEORY
• - an approach to link chemical kinetics to
thermodynamics
• - in more sophisticated versions: an approach
to understand the intermediates in a reaction
• - in reality there are many intermediates or
transition states, but we will lump them
together: they occur at low concentrations and
are very short lived
The rate of the overall reaction depends on the activation energy (G#)

G# = H# - T S#

(-G#/T)
The rate constant is proportional to: e

The function of a catalyst is to lower the activation energy

An enzyme is a highly specific catalyst for a given reaction

 TRANSITION STATE THEORY

A C A C A C

B D B D B D

An enzyme serves in several ways:

it binds and aligns the substrates at the active site
it stabilizes the transition state
it facilitates the redistribution of electrons (charges)
it promotes the breaking and making of bonds
 G ‘o

The enzyme does not alter the G ‘o

The enzyme chymotrypsin with its
active site and its substrate (black)
actually, there is a second substrate:
H 2O
Dihydrofolate reductase
with the substrates
tetrahydrofolate (yellow)
and NADP + (red)
 CHAPTER 6
Enzymes and Enzyme Kinetics
Michaelis-Menten (1912-1916)
k1 k2
E + S ES Products
k-1 + E
 CHAPTER 6
Enzymes and Enzyme Kinetics
k1 k2
E + S ES Products
k-1 +

E
 Derivation of Michaelis-Menten
equation

• 1. equilibrium assumption: k-1 >> k2

• 2. steady state assumption: d[ES]/dt = 0

Hmmm..??
Very short time interval Time interval during which
[ES] is at steady state

Garrett & Grisham

Biochemistry, 3rd Ed.
Fig. 13-08, p.413
Derivation of Michaelis Menten Equation from Wikipedia
 NB: [E] = [Eo] - [ES]

in past lecture(s) : [Eo] = [E]T, the total enzyme concentration at t = 0

Rearranging gives:

                            
                            
                      
The rate (or velocity) of the reaction is:
                  

Substituting and multiplying numerator and denominator by [S]:

                                                   
 k2 [Eo] [S]
x x

We use v , the initial velocity/rate, because we know [S]

vobefore
= much-------------------------
o
precisely only at the beginning of the reaction,
of the substrate has been used up
Km + [S]

when [S] >> Km

then

v  vmax and vmax = k 2 [Eo]

 k -1 + k 2
Km =
k1
 d

Km Kd
when

k -1 >> k 2
End of Lecture 3
• October 2, 2013