LM2 enzymes pt2 Flashcards
define uncompetitive inhibition
binds to the ESC preventing release of products
define competitive inhibition
binds to the active site preventing substrate binding
define non-competitive inhibtion
binds to the allosteric site, changing the shape of the active site so the substrate can no longer bond
how do you calculate velocity of enzyme activity
-d[s]/t
d[p]/t
define 1st order kinetics
linear increase in velocity with increase in substrate concentration
define 0 order kinetics
at plateau where there is no change in velocity with changing substrate concentration
what equation can be used to explain first order kinetics
y=mx+c
y= velocity
m= gradient
x=substrate concentration
c= intercept
what does the michaelis menten equation explain
explains the plateua mathematically and the aim is to establish a mathematical connection between V0,Vmax and Km
what is the dissociation constant / how do wwe find it
k1[E][S] = K-1[ES]
[E][S]/[ES] = K1/K-1
=Kd
what are the assumptions made about the enzyme reations
that it is in equilibrium and the concentration of ES remains constant during the enzymatic reaction therefore,
[ES] formation = [ES] breakdown
how do we get the Michaelis Menten constant
[E][S]/[ES] = (K-1+Kcat)/K1 = Km = MM constant
how can total enzyme concentration be given
E0=E+ES
what does Vmax equal
Vmax = Kcat[E0] when E0 = ES
What does V0 equal
V0=Kcat[ES]
what is the MM equation
V0 = Vmax[S]/Km+[S]
what do we assume when the substrate concentration is very large
when S»Km the Km can be ignored so the equation become V0=Vmax
how do we get a lineweaver burke plot
by inverting the michaelis menten equation to produce a straight line graph
y=1/V0
m=Km/Vmax
x=[S]
c=1/Vmax
what are the limitations of lineweaver burke plot
1/[S] and 1/V0 are small values and finding them on graph paper may be difficult
what is y=mx+c on a lineweaver burke plot
y=1/V0
m=Km/Vmax
x=[S]
c=1/Vmax
what does the lineweaver burke plot of a competitive inhibitor look like compared to without an inhibitor
there is a slope change so Km increases but y-intercept remains the same so Vmax is unaffected
they cross(inhibitor has steeper gradient)
what does the lineweaver burke plot of an uncompetitive inhibitor look like compared to without an inhibitor
Km and Vmax are reduced
runs parallel to the original
what does the lineweaver burke plot of a non-competitive inhibitor look like compared to without an inhibitor
Km remains the same but Vmax is reduced
both start at the same point but it has steeper gradient with inhibitor
what is the purpose of serine in the catalytic triad
acts as a nucleophile attacking the carbon in the carbonyl of the peptide bond
what are the amino acids in the catalytic triad
Asp102
his52
ser195
how do aspartate and histidine work together
the side chain of serine is an alcohol which is not a very good nucleophile so therefore the negatively charged side chain of aspartate interacts with the slightly positive charged hydrogen on the side chain of the histidine and will form a hydrogen bond, so the nitrogen on the histidine is now partially negatively charge and will interact with the alcohol group on serine transforming it to an alkoxyl group
describe the action of the serine alkoxide
it acts as a nucleophile and attacks the carbonyls carbon breaking the pi bond and forming a relatively unstable tetrahedral intermediate with a negative charge on the carbonyls oxygen
what is the purpose of the oxyanion hole
interacts with negative charge on carbonyls oxygen stabilising the tetrahedral intermediate
what does the tetrahedral intermediate collapse into
an amin and a carbonyl group
what happens after the tetrahedral intermediate collapses
the serine is acylated and forms an amide molecule that deprotonates the histidines nitrogen
what happens after the amide product departs
it departs with the hydrogen from histidine and histidine helps to transform water into hydroxide which attacks the carbon in a nucleophilic fashion creating another unstable intermediate to be stabilised by the oxyanion hole
describe the end products of the catalytic triad
a carboxylic acid product
all of the catalytic triad residues interacting with one another and the cycle repeats
define scissile bond
cleavage site
what polypeptide has a catalytic triad
chymotrypsin
subtilisin - similar to chymotrypsin but different residue numbers and slightly different arranged oxyanion hole
carboxypeptidase - different structures to enzymes above but same catalytic triad mechanism and also oxyanion hole
what would be proof of convergent evolution
all three enzymes evolved differently yet have similar catalytic triads and mechanisms
chymotrypsin
subtilisin - similar to chymotrypsin but different residue numbers and slightly different arranged oxyanion hole
carboxypeptidase - different structures to enzymes above but same catalytic triad mechanism and also oxyanion hole
what enzymes use other approaches that dont use activated serine residues
cysteine proteases
aspartyl proteases
metalloproteases
describe how cysteine proteases cleave a peptide bond
papain
uses a histidine activated cysteine residue to generate a nucleophile that attacks the peptide carbonyl
describe how aspartyl proteases cleave a peptide bond
the central feature of the active site is a pair of aspartic acid residues that act together to allow a water molecule to attack the peptide bond
one aspartic residue in its deprotonated form activates the attacking water molecule by poising it for deprotonation
the other protonated aspartic residue polarizes the peptides carbonyl group so it is more susceptible to attack
for example renin
how do metalloproteases cleave a peptide bond
the active site of such protein consists of a bound metal ion, almost always zinc, that activates a water molecule to act as a nucleophile to attack the peptide carbonyl group
describe the action of carbonic anhydrase
catalyses the rapid interconversion of carbon dioxide and water, carbonic acid and the bicarbonate ion and H+
describe structure of carbonic anhydrase
it contains a bound Zn2+ ion to the imidazole rings of three histidine residues as well as to a water molecule it is also in a cleft near the centre of the enzyme, the three histidines bind the zinc ion while one acts a base
what are the optimum conditions for carbonic anhydrase and how are they achieved
maximally active at a high pH
the binding of the water molecule to the zinc reduces the pKa of water from 15.7 to 7
with pKa lowered the concentration of OH- increases
what is the nucleophile in carbonic anhydrase
the zinc bound hydroxyl and is available to attack CO2 more efficiently than water
describe the action of the nucleophile in carbonic anhydrase
the zinc ion facilitates the release of a proton from water generating hydroxyl ions
the CO2 binds at the active site and is positioned next to the hydroxyl
the hydroxyl group attack the CO2 converting it to a bound bicarbonate ion
the catalytic site is regenerated with the release of HCO3- and the bonding of another water molecule
thus the binding of a water molecule to the zinc ion favours the formation of the transition state by facilitating the release of a proton and positioning the water close to the other reactant
what is the paradox associated with carbonic anhydrase
CO2 is hydrated at a faster rate than expected
what is the answer to our paradox
buffer concentrations above 10^-3 may be high enough to support the faster than expected rates of CO2 hydration
what is a histidine proton shuffle
his64 abstracts a proton from the zinc bound water thus generating nucleophilic hydroxide ion then buffer removes H+and deprotonates/regenerates His64
describe nucleoside monophosphate kinases
these enzymes catalyse the transfer of the terminal phosphoryl group from a nucleoside triphosphate usually ATP to NMP
what is the challenge of NMK
promote the transfer without promoting the competing reaction
what does the NMK domain consist of
central beta pleated sheet surrounded on both side by alpha helices as well as a key loop
what amino acid sequence does the p loop have
GLY-XXXX-GLY -LYS
why is it called the p loop
because it interacts with phosphoryl group on bound nucleotides
what do NMK require and why
they are basically inactive in the absence of a divalent metal ion such as Mg2+ which coordinates to ATP and fixes it in a particular conformation for tighter binding with the enzyme
what else does the divalent metal ion do for nucleoside monophosphate kinases
provides additional points of interaction between the ATP-Mg2+ complex and the enzyme thus increasing the binding energy
What does ATP binding do
induces large conformation changes and the p loop closes down on the polyphosphate chain interacting most extensively with the beta phosphoryl group and this movement brings down the top domain of the enzyme to form a lid over the bound nucleotide
what do both sets of changes during ATP binding ensure
catalytically component conformation is formed only when both the donor and acceptor are bound preventing wasteful transfer of the phosphoryl group to water
How do enzymes lower activation energy?
Orientating themselves to reduce the energy barrier associated with random movements rotations and vibrations
Enzymes can induce strain on a substrate stretching them and putting them into an unstable transition state
Enzymes can also temporarily add chemical groups to substrates and change their charge
