Biochemistry (I)

Lecture 10
Enzyme kinetics
• How to use michaelis menten equation
• What does Km and kcat mean?
• How to determine Km and kcat?
• Three types of reversible inhibition, competitive, uncompetitive, mixed, changes the
enzymes kinetic behavior

1
 Michaelis-Menten Equation

V max [S]
vo =
K M + [S]

V max = k cat [E] t

Where Km and kcat are constants at

given condition.

They are subjected to change with

change in temperature, pH, etc…
 How to experimentally construct Michaelis menten plot
• Using the same amount of enzymes, we can run and monitor the reaction with different
starting substrates.

• Then calculate the initial rate (ΔS/ Δt) and plot it onto a Rate vs. [S] graph

200 mM
 Important meaning of the terms describing enzyme kinetics

Term Description units

Vo Initial rate of reaction at given substrate concentration mol/L/s

Vmax Maximum velocity of reaction at given enzyme mol/L/s

concentration
Km Michaelis constant mol/L
• concentration of S needed to reach ½ Vmax
• Frequently associated with enzyme’s affinity for the
substrate
kcat Turnover number 1/s
• How fast this enzyme can react the substrate
kcat/Km Catalytic efficiency 1/(s*mol/L)
• Overall efficiency of an enzyme for reacting a
substrate

• kcat/Km has an upper limit of around 108 to 109, which is at the limit of
diffusion
 How to calculate kcat and Km?
• Currently, with the advance of computing technology, we can use curve fitting software that
fit the experimental data to Michaelis-Menten equation.

• Before we have that software, we can rearrange Michaelis-menten plot into linear form.
Most commonly, we use a double reciprocal plot (or frequently called Lineweaver-Burk plot)

Inverse on both sides…

Lineweaver-Burk Plot (double reciprocal)
 Some common enzymes and their Km & kcat

• kcat/Km varies greatly between different enzyme-catalyzed reactions

• Higher kcat/Km = more efficient (“better”) enzyme!

 Substrate specificity

• Same enzyme can have very different kcat/Km

values for different substrates

• The observed effects of an amino acid

mutation in an enzyme active site on KM and
kcat can be used to identify the role of the
amino acid in substrate binding (KM effects)
and transition-state stabilization (kcat effects).
B. G. Miller and R. Wolfenden, Annu. Rev. Biochem. 2002, 71:847-85
 t1/2 = 10-8 seconds
ODC SPA FUM STN MRA AMN CPB OSBS ADA CPA CDA ACE ATD CPA PTS KSI TIM CMU CYC CAN
10
log kdiff

5
109
0

log k
kcat/Km
-5
1023 kcat
kuncat
t1/2 = 5 seconds
-10

-15

t1/2 = 78 million years

-20

ADC, arginine decarboxylase; ODC, OMP decarboxylase; FUM, fumarase; SPA, sweet potato b-amylase; STN, staphylococcal nuclease; MRA,
mandelate racemase; CPB, carboxypeptidase B; ADA, adenosine deaminase; AMN, AMP nucleosidase; OSBS, o-succinylbenzoate synthase; CDA,
cytidine deaminase; PTS, phosphotriesterase; CPA(O), carboxypeptidase A for ester hydrolysis; KSI, ketosteroid isomerase; CPA(N),
carboxypeptidase A for amide hydrolysis; ATD, ascite tumor peptidase; TIM, triosephosphate isomerase; ACE, angiotensin-coverting enzyme;
CMU, chorismate mutase; CAN, carbonic anhydrase; CYC, cyclophilin.
B. G. Miller and R. Wolfenden, Annu. Rev. Biochem. 2002, 71:847-85
Enzyme Inhibition
 Enzyme inhibition

• Enzyme inhibitors are molecules that interfere with catalysis, slowing or

stopping enzymatic reaction.

• Two types of enzyme inhibition:

• Reversible: usually formed by non-covalent interaction between an
inhibitor molecule with enzyme

• Irreversible: a covalent attachment between inhibitor molecule and the

enzyme, permanently inactivates the enzyme.

• Among the reversible inhibition, there are three types of mechanism:

1. Competitive

2. Uncompetitive

3. Mixed
Competitive inhibition
 Competitive inhibition

• Because inhibitor “competes” with the substrate for the same active site, it will make
Km, or in this case we call apparent Km (Kmapp), larger.

• Vmax does not change

No inhibitor
Kmapp
+ inhibitor

α = factor to increase Km
[I] = concentration of inhibitor
Ki = dissociation constant for
inhibitor binding
 Competitive inhibition

• To analyze the inhibition, we will make the double reciprocal graph

• The lines cross at the same 1/Vmax, which shows Vmax is unchanged. We can use this
feature to indicate that this inhibitor is competitive

+ inhibitor

No inhibitor
 Competitive inhibition – how to determine Ki

• Lower the Ki, stronger the inhibition effect

• To determine Ki, we can experimentally construct the enzyme kinetic curves

with different concentrations of Inhibitor.

• Each point on this graph represents a

enzyme kinetic curve.
 Examples of competitive inhibition

• Competitive inhibitors are typically

substrate analogues
 Inhibitors as a drug target

• Transition state analogues mimcks the transition state of a substrate during enzyme reaction,
but cannot be reacted.

• Recall that enzymes bind transition very tightly. Therefore, by binding a transition state
analogue to an enzyme, enzyme cannot be do its normal function.

• This property can be used for drug design target

Oseltamivir
Inhibitor for neuraminidase –
which cleaves sialic acid
 Uncompetitive inhibition

• Uncompetitive binds to the enzyme-substrate complex (ES), and slows its Vmax or stops its
reaction.
Uncompetitive inhibition
 Uncompetitive inhibition
• Uncompetitive inhibitions changes both Vmax and Km.

• Vmaxapp = Vmax/α’

• Kmapp = Km/α’

• Double reciprocal plot gives parallel lines

 Mixed inhibition
• The inhibitor binds at a site on the enzyme surface different from that of the substrate.

• In this simplified example, the inhibitor binds to both free enzyme and the ES complex.
• EI has reduced substrate binding affinity compared to free enzyme.
• The EIS complex cannot carry out the catalytic event.
Mixed inhibition
Mixed inhibition
Reversible inhibition effects on apparent Vmax and Km
Reversible inhibition effects on Michaelis Menten Equation
Enzyme regulations

• While cells have transcriptional and translational controls over gene

expression and protein translation, it is also necessary to have protein
level regulation to ensure proper balance of cellular resources.

• Substrate level control:

• Product of a reaction may serve as an inhibitor to its enzyme

• Feedback controls:
• Metabolites downstream of a pathway can inhibit upstream
enzymes of the same metabolic pathway
Feedback controls

• Most frequently, negative feedback controls are used to control flow of

metabolites inside a cell

• Negative feedback (product feedback inhibition): product of a

pathway inhibits its upstream

• Activation feedback: product of a pathway may activate some

other pathways

• https://www.youtube.com/watch?v=DHZtOKyMPRY
How are downstream products able to control
upstream enzymes?
• Allosteric regulations – interactions between enzyme and a molecule that
induces enzyme to undergo conformational change.

• Conformational change may increase enzyme activity or

decrease.
• Example: ACTase

• ATP is an activator.
• CTP is an inhibitor.
Enzyme regulations: covalent modifications
 Reversible covalent modification by kinases/phosphatases:

•The target residues for ATP-dependent

phosphorylation by kinases are serine,
threonine, or tyrosine.

•The phosphoprotein is
dephosphorylated by a phosphatase-
catalyzed hydrolysis reaction.
Drugs developed as enzymes inhibitors
 Influenza virus
• The structure of the influenza
virus:

• The 13,600-nucleotide RNA

genome is packaged within
the sphere, about 120 nm in
diameter.

• The spikes on the virion

exterior include the
hemagglutinin molecule and
a spike that terminates in four
neuraminidase molecules.

• Hemagglutinin binds to N-acetylneuraminic

acid (Sialic acid) commonly found in cell
surface glycoproteins and/or glycolipids.

• At the end of infection cycle, Influenza virus

uses neutraminidase to cleave sialic acid in
order to release itself from the cell.
 Influenza virus

• Based on the crystal structure of

neuramidase complexed with sialic acid,
structural analogs of sialic acid were
developed with the potential to inhibit
the enzyme.

• Once neuramidase is inhibited, viral

particles cannot leave the infected cell.

• Partial model of the neuraminidase-

zanamivir complex, showing amino
acid residues that are close to the
binding site for the inhibitor.

• Oseltamivir (marketed as Tamiflu) was

used to treat influenza outbreaks in
2009 & 2013)
HMG-CoA Reductase inhibitor as cholesterol lowering drugs

• Once the rate-limiting role of HMG-CoA reductase in cholesterol

biosynthesis was understood, specific inhibitors were sought to
lower blood cholesterol levels.

• The compounds discovered are collectively called statins.

• They act by competitively inhibiting HMG-CoA reductase.

• Lovastatin and simvastatin are fungal polyketides and

atorvastatin (Lipitor® ) is synthetic.

• Each statin carries a mevalonate-like moiety (blue), explaining the

competitive nature of their activity.

• Inhibition of HMG-CoA reductase depresses de novo cholesterol

biosynthesis and, hence, intracellular cholesterol levels.

• This in turn leads to increased production of LDL receptors,

allowing more rapid clearance of extracellular cholesterol from the
blood, thus lowering blood cholesterol levels.
Biosynthesis of DMAPP & IPP – Mevalonate pathway
TCA cycle

HMG-CoA
reductase
ketone body biosynthesis

PMD ATP
Squalene synthesized from 2 FPP

3 DMAPP/IPP are used to make FPP (C15),

Then two FPP react and make squalene (C30)
Cholesterol biosynthesis
 Structural polysaccharides in bacterial cell surface

• On the outside of bacteria cell surface, there is a layer of peptidoglycan (polysaccharide-

peptide complex). Gram positive bacteria is shown below:

N-Acetylmuramic acid
N-Acetylglucosamine

Peptidoglycan layer has large crosslink network

 Formation of peptidoglycan
How do we get UDP-NAM?

In every step of adding an amino acid. What is

a likely co-reactant not shown here?
 Penicillin

• Penicillin works by binding to the

peptide chain, inhibiting its cross linking
with the other chains

• Cross-links between
adjacent peptidoglycan
chains are formed by the
action of a
transpeptidase enzyme.

• Penicillin, a structural
analog of the natural
substrate, reacts with
the active form of the
enzyme to form an
inactive covalent
complex that resembles
the enzyme–substrate
complex.