Measurements and calculations

• The standard Michaelis-Menten formulation is

v0=f([S]), but it’s not linear in [S].
• We seek linearization of the M-M equation so
that we can find Km and kcat, and so that we can
understand how various changes affect the
reaction.
• Lineweaver-Burk Plot
• Eadie-Hofstee Plot
Lineweaver-Burk Plot
• For linearization of the Michaelis-Menten
equation, take reciprocals on both sides:

V0 = Vmax[S]/(Km+[S])

1/V0 = (Km +[S])/(Vmax[S])

= (Km /(Vmax[S])) + [S]/(Vmax[S]))
= (Km/Vmax)*1/[S] + 1/Vmax
• Thus a plot of 1/[S] as the independent variable
vs. 1/V0 as the dependent variable will be linear
with Y-intercept = 1/Vmax and slope Km/Vmax
• Y-intercept is useful
directly:
compute Vmax = 1/(Y-
intercept)
• We can get Km/Vmax from
slope and then use our
knowledge of Vmax to get
Km; or
• X intercept = -1/ Km
… that gets it for us
directly!
Advantages and disadvantages of L-B plots
• Easy conceptual reading of Km and Vmax (but remember to take the reciprocals!)
• Suboptimal error analysis
• [S] and V0 values have errors i.e. not estimated accurately
• Error propagation can lead to significant uncertainty in Km (and Vmax)
Enzyme Inhibition
• Rates of enzyme reactions are often affected by the presence of
various chemicals and ions.
• Enzyme inhibitors combine, either reversibly or irreversibly, with
enzymes and cause a decrease in enzyme activity.
• Effectors control enzyme reactions by combining with the regulatory
site(s) of enzymes.
 Competitive Inhibition
• An inhibitor competes with a substrate
for the binding site of an enzyme
therefore it increases the KM of the
enzyme.
• enzyme–inhibitor complex does not
contribute to product formation, it
decreases the rate of product formation.
• competitive inhibitors have steric
structures similar to substrates, and are
referred to as substrate analogues. We
need to add more substrate to reach the
desired KM.
Lineweaver-Burk Plot for competitive
inhibition
• Km goes up so -1/ Km moves toward origin
• Vmax unchanged so Y intercept unchanged
 Non-competitive inhibitor

·inhibitor binds distal to active

site
·effects enzyme rate not affinity
i.e. Vmax is affected
·Reversible
·Lineweaver Burk intersect at
the Y axis
L-B for non-competitive inhibitors
• Decrease in Vmax  1/Vmax is larger
• X-intercept unaffected
Mixed Inhibition
• Inhibitor binds to enzyme
site that involves both S
binding and catalysis

• binds E in E S or E
Uncompetitive Inhibition
• binds covalently in the
transition state
• suicide inhibitor
• binds to the ES complex
• lowers affinity and velocity
• line-weaver Burke plots are
parallel
L-B for uncompetitives
• Km moves toward origin
• Vmax moves away from the origin
• Slope ( Km/Vmax) is unchanged
Penicillin as a suicide substrate
• suicide substrates are often un competitive inhibitors that decrease the
energy of the transition state and allow the ES to have lower energy
that that of the EP.

• Bacterial cell wall - extensive cross linking of sugars and peptides

• Penicillin (and ampicillin) have a highly reactive ß lactam ring which

makes a peptide bond very reactive.
• Penicillin mimics the peptide alanine residues and forms a low energy
intermediate by covalently reacting with a serine

• In molecular biology, we use this as a tool. Ampicillin will stop E.

coli growth. Bacteria that have a gene (plasmid) inserted into the
bacteria have ß lactamase. An enzyme that hydrolyses the reactive
peptide bond found in amicillin and penicillin
 Competitive Noncompetitive Uncompetitive

Binds active site binds to other than Transition analog

binding site
inhibition reversed binds covalently
by increasing [S] not reversed by ES not E free
increasing
Kmapp increases with changes both x and
inhibitor (x axis no effect on S y axis (Km and Vmax)
intercept changes) binding (Km) only
slows down rate
no change in 1/Vmax (V)

Usually analogs of decreased Vmaxapp

substrate (Y axis intercept)

inhibitor binds
both E free and ES
complex
• An organism must be able to regulate the catalytic activities of its component
enzymes

• coordinate many metabolic processes

• Respond to changes in the environment

• Growth and differentiation

• Both Inhibitors and affectors

• do not follow Michaelis-
Menten kinetics - instead use a
hill plot for both + and –
effects

• similar to O2 dissociation of
hemoglobin