BIOC 503 - General Enzymology II

kinetics

study of the rate at which compounds react

Affected by:

• enzyme 

• substrate 

• effectors

• temperatures

Reasons to study kinetics

• quantitative description of biocatalysis

• determine the order of binding of substrates

• understand catalytic mechanism

• Find effective inhibitors

• understand regulation activity

k1

rate of ES complex formation from substrate and enzyme

k-1

rate of disassembly of ES complex back to enzyme and substrate

k2

aka kcat because it is the rate of the catalytic reaction forming the product and returning free enzyme

• slowest reaction

• rate limiting step

k-2

formation of ES complex from enzyme and product

• has a value of about 0 at V0

steady state approximation

enzyme is saturated with substrate ([S]»[E]) therefore [ES] is constant as rate of ES complex formation and breakdown are equivalent.

free ligand approximation

[S free] = [S total] because so much substrate compared to the amount of enzyme.

rapid equilibrium approximation

k2 =kcat << k1 and k-1 so equilibrium is reached quickly

saturation kinetics approximation

k2 is rate limiting so Vmax = k2*[Et]

greater

when [S] is much ___ than Km, then Km is negligeable to [S]

V0

__ = (Vmax*[S])/(Km+[S])

turnover number

k2 or kcat is also called the ___ aka how many substrate molecules one enzyme can convert per second

Michaelis-Menten constant

Km is the ____ aka an approximate measure of substrate’s affinity for an enzyme

presence of product, absence of substrate

To get initial velocity values, you measure either the ____ (curve going up) or the ____ (curve coming down) usually via absorbance against time.

Michaelis-Menten kinetics

ideal rate refers to the ____ aka V = (Vmax*[S])/(Km+[S])

deviations

__from ideal rates can be due to

• limitation of measurements (dead time before measurement starts can be significant)

• substrate inhibition

• substrate prep containing inhibitors (not common modern day because everything pretty pure)

• enzyme prep containing inhibitors (not fully purified out of solution)

1/2Vmax

Km = ____

y axis Lineweaver Burk-plot

1/v

x axis Lineweaver Burk-plot

1/[S]

y-intercept

1/Vmax

x-intercept

-1/Km

Lineweaver Burk plot

linearized double reciprocal plot analysis

• determine Vmax and Km

• determine 2 substrates mechanism

• determine inhibition type

Km/Vmax

slope of Lineweaver Burk plot = ___

enzyme specificity

= Kcat/Km

• limited by diffusion from active site

velocity, affinity

enzymes can get more efficiency by having high ___ (aka makes product faster, bigger kcat) OR by having higher affinity (binds substrate faster)

koff/kon

Km = ___ = (k-1 + k2)/k1 = (breakdown/formation)

sequential mechanisms

two substrates reactions

E —> S1 —> S2 —>P +E

substrate one binds, then substrate 2 can bind, then products are made

• LINES INTERSECTING TO THE LEFT OF Y-AXIS ON LINEWEAVER BURK PLOT

ping-pong mechanism

two substrates reactions

E—>S1 —> P1 + E’ —> S2 —> P2 +E

substrate one binds, modify enzyme and first product released, substrate 2 bonds to modified enzyme, product 2 is release and enzyme back to original form

• LINES PARALLEL ON LINEWEAVER BURK PLOT

irreversible

___ inhibitors REACT with the enzyme

• one molecule can permanently shut off enzyme

• often powerful toxins but also some drugs

reversible

__ inhibitors bind & dissociate from enzyme

• often structural analogs

• may be used as drugs to slow down specific enzyme

• prevent substrate binding OR prevent the reaction

competitive inhibition

binds to the same active site as the substrate 

• does NOT affect catalysis

• Vmax stays the same

• Km increases

• Lineweaver Burk plot: all lines intersect at the y-axis

uncompetitive inhibition

• only binds to ES complex

• inhibits catalytic function

• Vmax decreases

• Km decreases

• Lineweaver Burk plot: lines are parallel & NO CHNAGE IN SLOPE

mixed inhibition

• binds enzyme with or without substrate

• inhibits both substrate binding and catalysis

• Vmax decreases

• Km changes

• Lineweaver Burk plot: line intersect left of y-axis

noncompetitive

__ inhibitors are mixed inhibitors such that there is NO CHANGE IN Km

enzyme activity regulation

• noncovalent modulation (allosteric)

• covalent modification

• irreversible

• reversible

allosteric regulators

• binds to secondary sites

• generally small chemicals

• either positive (improve enzyme activity) or negative (decrease enzyme activity)'

• do NOT follow Michaelis-Menten kinetics

reversible covalent modifications

• phosphorylation

• acetylation

• ubiquitination

• methylation

• adenylation