B2 mechanisms of enzyme catalysis Flashcards
what is the essence of catalysis
the specific stabilisation of the transition state
what do catalysts do
-lower the activation energy
-accelerator of a chemical reaction
-increase the rate of rxn
-is not consumed in the rxn
-does not affect the eqm
how is the active site of an enzyme formed
folding of protein brings side chains of various aa that may be far apart in the primary seq into close juxtaposition
how many steps are in the enzyme-catalysed reaction
3
enz+S– enz-S–enz-P–enz+p
what allows the reaction to occur in regards to active site and substrate
positioning of the substrate mols in the most favourable relative orientation for the rxn to occur
the active site is perfectly ??? to the transition state
complementary
what do the aa side chains of the active site do to the electron distribution of the transition state
stabilise
the substrate is ??? on binding to the active site
strained
enzymes do what to the activation energy and reaction rate
lowers the activation energy
increases the reaction rate
what are the non covalent interactions between the substrate and the aa side chains of the enzyme
-acidic groups (Asp, Glu)= ionic bonds
-basic groups (Lys, His, Arg)= ionic bonds
-hydrophillic ints woth -OH or alc groups (Ser, Thr, Tyr)
-hydrophilic ints with -SH or thiol groups (Cys)
-hydrophilic ints with amide groups (Asm, Gln)
-aromatic ints (Phe, Tyr, Trp)
-hydrophobic ints (Ala, Leu, Lle, Val, Met)
how do reactive groups at the catalytic site surface catalyse the rxn by
-donating or withdrawing electrons
-stabilising or generating free radical intermediates
-forming temporary covalent bonds
(transition state intermediate)
what are cofactors
non protein molecules in addition to enzymes
3 examples of cofactors
-metal group (hexokinase Mg2+)
-prosthetic group
covalently bound organic mol (heme, lipoic acid)
-coenzyme
tightly but not covalently bound organic mol (NAD)
name of an enzyme protein WITH prosthetic group/coenzyme and is it catalytically active
holo-enzyme
catalytically active
name of an enzyme protein WITHOUT prosthetic group/coenzyme and is it catalytically active
apoenzyme
catalytically inactive
what does specificity mean
enzymes catalyse only one specific rxn
oxidoreductases (general)
oxidation and reduction reactions
dehydrogenases
addition or removal of H
oxidases
2 electron transfer of O2 forming H2O2
2 electron transer to 1/2 O2 forming H2O
oxygenases
incorporate O2 into product
hydroxlases
incorporate 1/2 O2 into product as -OH and form H2O
peroxidases
use as H2O2 as oxygen donor, forming H2O
transferases (general)
transfer a chemical group from one substrate to another
kinases
transfer phosphate from ATP onto substrate
hydrolases
hydrolysis of C-O, C-N, O-P, and C-S bonds
examples of hydrolases
esterases, proteases, phosphatases, thioesterases
lyases
addition across a carbon-carbon double bond
examples of lyases
dehydratases, hydratases, decarboxylases
isomerases
intramolecular rearrangements
synthetases
formation of bonds between two substrates
frequently linked to utilisation of ATP
units of catalytic/enzyme activity
number of micromoles (umol) of substrate converted per minute under standard optimised conditions at 30 degrees
1 enzyme unit (EU)= 1umol min-1
what is specific activity
activity of an enzyme per milligram (mg) of total protein in the enzyme prep
(expressed in umol min-1mg-1)
what does specific activity give a measurement of
purity of the enzyme
why is the rxn rate hyperbolic
accumulation of product
depletion of substrate
denaturation of enzyme
requirements of measuring the rxn rate
measured at fixed enzyme conc
defined temp and pH
for meaningful quantitative assays of enzyme activity it is necessary to ensure what
that initial velocities (V0; rxn rates) are measured
5 factors that affect enzyme activity
-pH
-temperature
-concentration of enzyme
-concentration of substrate
-covalent modification of enzyme
what is pH and its parameters
a measure of the acidity of alkalinity of a solution
acidic < 7.00 < basic
neutral pH = 7.00
what of the aa side chains depends on the pH of the sol
ionisation state
what is dependent on the pH of an enzyme catalysed rxn
binding of the substrate and catalysis
do chemical reactions proceed faster or slower at higher temps and why
faster
mols move faster, greater chance to strike
electrons gain activation energy easier
what happens when an enzyme is denatured
loss of H bonding
unfolding
precipitation
loss of activity
the effect of varying the amount of enzyme
predictable linear increase in product formation with increasing amount of enzyme
what does the michaelis-menten eqn describe
the dependence of rate of rxn on concentration of substrate at steady state (ES formation balanced by its removal) and vast molar excess of substrate over enzyme [S]»[E]
what do the following in the michaelis menten eqn mean
v
Vmax
[S]
Km
v rate of rxn
Vmax maximal rate of rxn
[S] conc of substrate
Km Michaelis constant
what is the michaelis menten eqn
v= Vmax[S]/Km + [S]
what does high Km and low Kn correspond to in terms of substrate
high = low affinity
low = high affinity
enzymes with low Km compared with the conc of substrate [S] in the cell act at their ?
max rate
do modest changed in the conc of substrate [S] have an effect on the rate of rxn
no
effect of rate of rxn where enzymes with a high Km and a small change in [S] conc
large change
experimental determination of Km and Vmax
-incubation of the enzyme under optimal conditions for a short time
(assuming no change on conc of [S] or [product] is negligible compared with [S]
-using range of [S] concs
-plotting double reciprocal graph of rxn rate over [S] conc
-extrapolating back from experimental points to determine intercepts
what is the plot given to the double reciprocal plot
lineweaver-burk
1/rate
1/Vmax
-1/Km
1/[substrate]
what occurs in a sequential rxn
(enzyme with 2 substrates)
each substrate binds in turn
what is a ternary complex
and what do the lines look like on LB graph
complex containing three diff mols A-Enz-B
converging
dihydrofolate reductase enzyme pathway
dihydrofolate+NADPH+H+—-tetrahydrofolate+NADP+
ping pong rxn
(enzymes with 2 substrates)
and what do the lines look like on LB graph
one substrate reacts, and modifies enzyme
then second substrate reacts with modified enzyme
parallel lines
what is an allosteric enzyme and where are the often found
contain binding sites other “allo” than substrate binding sites
often in multi subunit complex
more than one active site in complex
what does the binding of a substrate to an allosteric enzyme lead to
binding to active site of first subunit leads to change in conformation facilitating binding of substrate to the other active site
what is a reversible inhibitor
non covalent (eqm) binding to enzyme
many are relatively unspecific
mechanism of a reversible inhibitor
blocking substrate binding or hindering catalytic steps
what are irreversible inhibitors
inactivators
bind to enzyme covalently (suicide inhibitor)
many are substrate analogues
undergo part of rxn
transition state covalent intermediate does not break down
what is a competitive inhibitor
competes with substrate for binding at the active site
is a function of the relative affinities of the substrate and the inhibitor for binding the enzyme
inhibition is a function of relative conc of substrate and inhibitor
effect of competitive reversible inhibition on michaelis menten and LB
Vmax is unchanged, Km increased
if enough substrate added, can overcome inhibitor
what are mixed inhibitors
they do not bind to the active site
inhibitor can bind prior to substrate or to the enzyme-substrate complex
mixed inhibitors distort the substrate binding site which affects
-apparent substrate affinity
-catalytic turnover (slowing catalysis)
effect of mixed inhibitors on Km and Vmax
either increase or decrease Km
decrease Vmax
binding position and competition of non competitive inhibitors
binds to the enzyme at a position separate from the active site
no competition for binding with the substrate
what happens to the affinity and rate of rxn with non competitive inhibitors
apparent affinity for the substrate is unchanged, but the rate of rxn is slowed
effect of non competitive inhibition on MM eqn
and effect of adding more [S]
Km is unchanged, Vmax is decreased
adding more substrate has no effect on the rate of rxn
effect of allosteric inhibitors on Km and apparent affinity of enzyme
increase the Km and lower to apparent affinity of enzyme for its substrate
