Lecture 8 - enzymology 2 Flashcards
how can enzymes be inhibited?
by reversible inhibition such as competitive, noncompetitive and uncompetitive or irreversible
why study enzyme inhibitiors?
enzymes make great target drugs
what are competitive inhibitors?
Competitive inhibitors are molecules that compete for the active site blocking substrate binding
The inhibitor is structurally and chemically similar to the substrate
As the inhibitor is in competition with the substrate, its effects can be reduced by increasing substrate concentration
Examples include:
Sildenafil (Viagra) PDE5 inhibitor, treatment of erectile dysfunction
Zanamivir (Relenza) neuraminidase inhibitor, treatment of influenza
The presence of competitive inhibitor reduces available enzyme active sites for the substrate.
Increased substrate concentrations required to reach enzyme saturation
Vmax can be attained with increasing concentrations of substrate
Competitive inhibitors change the apparent value for KM
KM apparent= KM(1 + [I]/Ki )
what are non competitive inhibitors?
Non-competitive inhibition involves a molecule binding to a site other than the active site (anallosteric site)
The binding of the inhibitor to the allosteric site causes a conformational change to the enzyme’s active site therefore, the substrate no longer share specificity, meaning the it cannot bind
As the inhibitor isnotin direct competition with the substrate, increasing substrate levels cannot mitigate the inhibitor’s effect
Examples include:
Cyanide: binds to cytochrome oxidase (complex IV) blocking the electron transport chain and the production of ATP via aerobic respiration
Non-competitive inhibitors bind to free enzyme and also to the ES complex reduction in concentration of active enzyme
It decreases the maximum reaction rate but has no effect on how quickly substrate saturation is achieved
Vmax cannot be attained with increasing concentrations of substrate
what is the mechanism and specificity of serine proteases?
Residue numbers here refer to chymotrypsin.
Di-isopropyl phosphofluoridate (DIPF) reacts with Ser195 to inactivate chymotrypsin.
The catalytic triad removes a proton from Ser195 and makes it a very strong nucleophile
Ser195 becomes covalently attached to the substrate in the acyl enzyme – covalent catalysis
The transition state makes favorable interactions with the oxyanion hole that neither substrate nor products can make – the transition state is stabilized
His57 acts as an acid and a base catalyst at different steps
The substrate-binding pocket structure determines substrate specificity
what is the turnover number?
how fast an enzyme can work or the number of molecules of substrate converted to product per second by one enzyme active site
v = k2[ES] k2 is also called kcat or turnover number
[S] excess At saturation all sites occupied: Vmax = kcat[Etotal],
kcat = Vmax / [Etotal], i.e the number of molecules of S converted to P by one enzyme active site at saturation with substrate per unit time (moles form per second)
what is an example of a turnover number - kcat
In an assay of chymotrypsin (Mr = 25000) containing 25 µg of the enzyme the
Vmax is 24 µmol min-1
Calculate the turnover number of chymotrypsin
25 µg = 25x10-6/25000 mol = 10-9 mol = 1 nmol chymotrypsin Calculation of the molar concentration of enzyme
Vmax / [Etotal] = 24x10-6 mol min-1 / 10-9 mol
kcat = 24.103 min-1 = 24000 min-1 = 400 s-1 –> 400 moles of product formed per second=turnover number
what are nerve gases?
Irreversible inhibitors usually react covalently with a group at the active site that is essential for catalysis
Sarin reacts with one Ser residue in acetylcholine esterase enzyme active site
Sarin and Novichok agents have been used as chemical weapon in many conflicts and act via similar mechanisms
Penicillin irreversibly inhibits the transpeptidase enzyme responsible for bacterial cell-wall synthesis
Sarin inactivates acetylcholine esterase at neuromuscular junctions
how are enzyme inhibitors used as drugs?
Statins are competitive inhibitors of HMGCoA reductase that lower cholesterol and are used in prevention or treatment of heart disease
More than 8 million people in the UK take statins every day.
Heart disease is particularly prevalent in the West of Scotland.
Lovastatin was the first statin to be marketed
what is the summary of this lecture?
Turnover number: the number of molecules of substrate converted to product per second by one enzyme active site
Inhibition of enzyme activity: visualization using Michaelis-Menten and Lineweaver-Burk plots
Irreversible inhibitors: catalysis blocked
Reversible inhibitors:
Competitive inhibitors: Vmax can be attained as [S] increases; no change in Vmax but change in Km
Non-competitive inhibitors: no change in Km but Vmax variable depending on [I], not altered by [S]
Serine proteases – catalytic triad, mechanisms of catalysis and specificity defined by structure of binding pocket
how do we visualise inhibition?
Michaelis-Menten plot. where you compare for the 2 inhibitors Vmax and Km
Visualising inhibition: Lineweaver-Burk plotCompetitive inhibitor
E + I → EI
A competitive inhibitor increases the Km value
There is no change in Vmax
the lines cross over in the y axis
Visualising inhibition: Lineweaver-Burk plotNon-Competitive inhibitor
The Vmax value is decreased in the presence of the inhibitor
There is no change in Km
the 2 lines begin at the same source and dont cross over
look at the lecture for how Values of Vmax and Km can be obtained from a Lineweaver-Burk plot
