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Fundamentals 10-11
8/27/08 – Enzymes II
*This transcript was a little more difficult to follow, due to the wonderful transparencies
along with Dr. Baggott enjoys to mumble! So please let me know if you have any
problems following or if you have any questions.
 [S] >>> [E]
o Two different curves: 1. Right hand hyperbolic curve 2. Sigmoid curve
that corresponds to an allosteric site.
Characteristics of Enzymes
 The active site is small surface area
 It is held together by polypeptide chain
 Amino acid residues catalyze the reaction, the side chains
 Active site is specific for one type of substrate
 Enzymes are usually specific for one type of reaction
 Catalytic properties are explained by how well it binds to its substrate
 Michaelis-Menten kinetics, the efficiency is determined by Vmax
 Affinity is measured by Km
Regulation of Enzymes
 Methods of regulation of existing amount of enzymes:
o Reversible Inhibition
o Inhibition or stimulation by other proteins (won’t discuss too much)
o Reversible covalent modification
o Zymogens or enzyme precursors
Allosteric Enzyme
 Will have sigmoid kinetics
 Means “other site,” enzyme has other sites for small molecules to regulate
 Almost all allosteric enzymes are oligomers, they can be dimmers, trimmers, etc.
 They are all big, in order to generate all sites for activation and/or inhibition
Ways in which we can modify activity of enzyme by covalent
1. Phosphorylation: mostly used by mother nature to modify the activity of an
enzyme. When phosphorylate, you either increase or decrease its Km or its
Vmax or sometimes both. Generally you increase or decrease its activity.
You can phosphorylate anything that has an –OH hanging from it; such as
Tyr, Thr, or Ser, and sometimes His.
2. Adenylation: Adding the elements of AMP, adding the ribose phosphate;
using ATP; usually occurs on a Tyr. The phenyl group is subject to many
mechanisms that covalently modify the enzyme, because of the –OH is a good
target to attach something.
3. Uridylation: Adding the elements of UMP.
4. ADP-ribosalyation: uses co-enzyme nicotine to add NAD, throws away the
vitamin portion of the enzyme and just uses the ADP. Used in DNA repair,
especially when you have a mutated base, it is taken out of DNA and a new
one is put in its place to repair DNA.
5. Methylation: deals with Glu
When you have an enzyme with an –OH group, such as Tyr, it can be
phosphorylated and you know that if the enzyme is phosphorylated, there will
be a way to take that phosphate group off.
A protein kinase will phosphorylate (puts on a phosphate on the enzyme); a
phosphatase will take off the phosphate and return to original form.
 Most common are used to digest proteins; they are produced in the pancreas;
secreted in the lumen by zymogen granules and then into the small intestine to
digest the protein.
 All are inactive. They are an enzyme precursor to an enzyme with very little
activity. – ogen, means it is a precursor.
 Why create enzyme with little activity? If you created active enzyme it would
begin to digest (or whatever the activity) and would be a bad thing.
 Ex. Chymotrypsinogen is cleaved and almost always cleaved at the polypeptide
portion to create chymotrypsin, which is now the active form.
 Almost all enzymes are active around a neutral pH of 7 to 8. That is because there
will be a base that will be unprotanated when the pH comes up and an acid that is
unprotanated as the acid comes up. There is a range where maximum activity of
enzymes is at physiological pH (7). Very few are active above pH of 9 or 10,
however some are active at lower pH such as pepsin, which is utilized to digest
proteins in the stomach where pH is around 2 or 3.
 Anaerobic glycolysis -> the reason why you can’t keep running -> produces lactate,
which is an acid. Producing lactate, drops the pH of your muscles of 7.2 to about 6.1.
When this pH drops the activity of the enzymes that are used to make ATP for your
muscles is decreased, and you cannot keep running.
Substrate “Channeling”
 In its best form during fatty acid biosynthesis
 Simply means the product of one enzyme catalyzed reaction is furnished to
another enzyme. It doesn’t diffuse into the bulk solution, it is furnished
nanometers away from the active site.
 You go from one active site to another active site without the products
diffused into the bulk solution.
 You have all kinds of intermediates in fatty acid biosynthesis
o Ex: fatty acid biosynthesis does make a intermediate = a C6 fatty
acid, which in never found inside cells, because it is furnished into
another fatty acid and then others, finally into C16 which will diffuse
away from the fatty acid biosynthetic pathway. None of the others
 Are oligomers where you have 2 or more different monomers involve
 The catalyze the same reaction, but may have different kinetic constants, like Km
and Vmax may be different.
 Useful in determing how much heart damage or liver damage has been made, by
looking at the amount of isozymes in the blood.
 Isozymes are normal, everyone has them; they are not a mutation just a different
type of enzyme.
 Genetic polymorphism -> means the amino acid sequence of your enzymes is not
the same as the amino acid sequence of my enzymes. – this won’t be on the test!Enzyme Kinetics!
 We will only be speaking about enzymes that obey the right handed hyperbola
behavior; not sigmoid.
 Michaelis-Menten equation/kinetics: predicts how an enzyme will act
o Vi = Vmax [S] / [S] + Km
There are 3 portions of the curve: (also refer to transparencies for graphs)
o [S] is very high relative to Km ; Vi = Vmax (0 order)
 [S] >>> Km
o [S] is very low relative to Km; Vi =Vmax [S] / Km (1st order)
 Km >>> [S]
He makes a point that on the graph the line is NOT a line, but a bunch of
points/experiments on a line.
Double reciprocal plot
 The reciprocal of the Michaelis-Menten equation
 You go from a hyperbolic plot to a straight line with a slope = Km/Vmax and a yintercept = 1/Vmax (refer to graphs on transparencies)
Michaelis-Menten equation – Assumptions and Conditions
 [S] >>> [E]
 [E] = constant
o E + S <---> ES - E + P
(reversible) - (irreversible)
 Steady state
o d[ES] / dt = 0 This means that the concentration of ES does not change.
o d[ES] / dt = 0 = k1 [E] [S] – k2 [ES] – k3 [ES]
o [E]total = [E] + [ES]
o [ES] = Etotal [S] / (k3 + k2)/ k1 + [S]
Measure initial velocities
o Vi = d[P] / dt = k3 [ES]
o Michaelis-Menten => Vi = k3 [ES] = Vmax [E]t [S] / Km + [S]
 For the test know the four assumptions and conditions noted above to
form the M-M equation.
Bisubstrate Reaction
 Most are 2 substrates to 2 products is most common.
 You can have a few different mechanisms that accomplish this bisubstrate
1. Sequential: all enzymes bind to substrates before any products are
released/produced. (refer to EAB complex drawing)
a. Binding can be random. A can bind first or B can bind first,
and then the next substrate will add after that one. You get to
the final complex before you produce products.
b. Binding can be ordered. One substrate HAS to bind first
before the other substrate can bind.
2. Non-sequential (“ping-pong”): Products are produced before all
substrates are bound.
Meaning of Km
 Units are in concentration = mM, uM, etc.
 k3 + k2 / k1 *k3, very frequently, will be a lot smaller than k2, therefore
changing the equation to:
o Km = k2/k1  In terms of equilibrium [E] [S] / [ES]
If Km is small, [ES] is large (higher affinity), and E & S will be small
If Km is high/large, [ES] is small, and E & S are high/large
o Think backwards!
o Ex: Km = 1 x 10^-6 A
Km = 1 x 10^-8 B
Km of B binds tighter
o Higher the Vmax the more efficient the enzyme will be.
3 Types of Inhibition
1) Competitive: (refer to graph) A competitive inhibitor is most common – it
changes the Km, not the Vmax. It increases the apparent Km and competes for
the same active site.
2) Pure Non-Competitive: (refer to graph) A pure non-competitive inhibitor is more
rare – it changes the Vmax.
3) Mixed Inhibitor: changes both the Vmax and Km.
Km – binding of substrate to enzyme
 Lower the Km, tighter the binding of substrate to the enzyme
Ki – I (inhibitor) binding to E (enzyme)
 Lower the Ki, tighter the binding
 Ex: I1
Ki = 1 x 10^-6
Ki = 1 x 10^-12
 Ki = [E] [I] / [EI]
I2, Ki binds tighter