Mechanisms Flashcards
electronegativity
ability to pull an electron increases bottom left difference = polarisation + dipoles i.e. in water partial +ve charge on H + partial -ve charge on the lone pair
nucleophiles
i.e. carbanion,
oxygen atom in an unprotonated hydroxyl group carboxyl group, negatively charged sulphur in a cysteine, nitrogen of a histidine side chain
electron rich w strong tendency to donate to an electron deficient atom
electrophiles
i.e. carbonyl carbon,, phosphorous of a phosphate group, proton
electron diffiecient accept electrons
general acid/base catalysis
lysine, arg
sys
his
ser
tyri
.e. in enolase
proton transfer to/from a molecule other than water
glutamic and aspartic - protonated form - acid catalysis
ionised form can pick up a proton - base catalysis
mimic transition states with analogs
hydroxyl ion attacking ester = diol intermediate with 1 O- attached to carbon before being cleaved
stable phosphonate ester analog allows us to visualise
carbonate hydrolysis
chymotrypsin - cleaves peptide bonds on C terminal side of pheylalanine
from pancreas
degradation of polypeptide chains
produced by synthetically inactive form which needs to be activated by other proteases
precursor chymotrysinogen clipped by trypsin between 15 and 16 = pi chymotrypsin
= active by attacks itself - autolysis
= released dipeptide of seriene 14 and arginine 15 = break in the chain +
removes residues threonine 147 and asparagine 148 = gap in the chain
structural adjustments in amino acid sequence enable fold of the enzyme = active conformation
beta sheets in the structure
characteristic active site - catalytic triad
disulphide bridge to stabilise structure
burst phase equiv. to 1 para nitrophenol produces per enzyme mol produced in reaction
chymotrypsin is covalently linked to the substrate during the catalytic cycle forming an acyl enzyme intermediate = para nitrophenylacetate
= enzyme cleaving an ester bond to release nitro phenol and eventually acetic acid
fast phase - release of intermediate para -nitrophenol easy to detect has a distinct colour
remainder of reaction
cleavage of acyl enzyme to produce acetic acid
slow phase is limited by burst phase at the beginning that fills up enzyme
chymotrypsin mechanism
substrate binds into the active site in pocket
carbonyl carbon projected towards oxyanion hole
serine side chain
attracting with the histidine = strong nucleophilic ion on side chain of serine 195
removing proton = attacks peptide carbonyl group = tetrahedral enzyme intermediate + short lived negative charge on carbonyl oxygen of the substrate which is
stabilised by hydrogen bonding within the oxyanion hole - by 2 NH groups 1 from seriene one from glycine residue
= short lived intermediate
and instability of the negative charge on the substrate carbonyl here = leads to collapse of tetrahedral intermediate w reformation of a double bond between carbonyl carbon and the oxygen - displaces bond between carbon and NH group of next amino acid along
breaking that bond + protonation of amino group
seriene residues now in a covalent bond with the N part of the substrate still in the oxyanion hole
hydrolysis of acyl-enzyme intermediate by a water molecule which is deprotonated by general base catalysis by a histidine residue = hydroxide ion a nucelophile which attacks the ester linkage through the carbon of the ester degrading the acyl enzyme intermediate utilising the oxyanion hole to carry the negative charge of the ester oxygen
leave the active site to give us back our seriene residue to the histidine
phenyl alanine ring N terminal side of the bond to be cleaved
active site of enzyme has a hydrophobic pocket where phenylalanine side chain sits = specificity of enzyme come from
active site catalytic triad is made up of a carboxylic acid, aspartic acid residue, histidine residue and a seriene residue -side chain of the seriene is hydrogen bonded to the histidine residue of the catalytic triad
oxyanion hole - where the NH on the seriene projects
step 1: the acylation phase
ES complex:
O- in oxyanion hole
covalent bond formed between serien and carbonyl carbon = break down of peptide bond
proton comes onto the NH as it leaves. the unstable intermediate with the negative charge on the oxygen degrades to put the double bond back. the product is able to leave. the other part of the substrate is in covalent linkage with serine 195. This is end of the fast burst phase
catalytic triad:
aspartic acid 102
histodine 57
seriene 195
when the substrate binds
the side chain of the residue adjacent to the peptide bond to be cleaved, nestles in a hydrophobic pocket on the enzyme, positioning the peptide bond for attack.
when serine and histodine interacts a strong nucelophilic alkoxide ion is generated on seriene. the ion attacks the peptide carbonyl group forming a tetrahedral acyl-enzyme. a short lived negative charge on the carbonyl oxygen of the substrate is stabilised by hydrogen boning to the oxyanion hole.
seriene
- the deacylation phase
the acylated enzyme has
an incoming water molecule is deprotonated by general base catalysis
hydrogen removed by the nitrogen on histodine 57 generating a hydroxide ion which is stongly nucleophilic.
it attacks the partial positive charge on carbonyl carbon
which is excentuated by the development of the negative charge on the oxygen of the carbonyl carbon which resides in the oxyanion hole = stabilised
this metastable tetrahedral intermediate collapses
and displaces seriene 195.
left with carboxylate anion product and disassociate from enzyme leaving seriene 195 intact and its side chain hydrogen bonded again with histodine
pH dependence of chyrotrypsin
histodine 57 is invovled in catalysis
@ low pH histidine becomes positively charged
kcat falls
at high pH the iceleucine N terminal becomes uncharged salt bridge is broken and Km increases.
Lysozyme
cleaves peptidoglycan carbohydrate component in bacterial cell walls that provides stability against osmotic pressure
polypeptide chains of copolymer of ecytyl glucosymeen and mur2
sugar chains linked by peptide crosslinks
hydrolyses Mur2 Ac-GLcNAc glycosidic bond
Glu35 and Asp52 are essential in active site for activity
Sn2 mechanism
bimolecular nucleophilic substitution reaction
i.e. bromoethane with hydroxide ion where bromine is displaced
initiation by Aspartic acid residue 52 - acting as a covalent catalyst displacing GLucNAc via an Sn2 mechanism. ASp52 attacks attacks carbon 1 on hexose in an Sn2 displacement reaction knows out other part of sugar chain from active site. requires GLu 35 protonates the GLucNAc =
Oh group departs coming from proton from Glu 35
there is a covalent intermediate form between
Lysozyme
cleaves peptidoglycan carbohydrate component in bacterial cell walls that provides stability against osmotic pressure
polypeptide chains of copolymer of ecytyl glucosymeen and mur2
sugar chains linked by peptide crosslinks
hydrolyses Mur2 Ac-GLcNAc glycosidic bond
Glu35 and Asp52 are essential in active site for activity
Sn2 mechanism
bimolecular nucleophilic substitution reaction
i.e. bromoethane with hydroxide ion where bromine is displaced
initiation by Aspartic acid residue 52 - acting as a covalent catalyst displacing GLucNAc via an Sn2 mechanism. ASp52 attacks attacks carbon 1 on hexose in an Sn2 displacement reaction knows out other part of sugar chain from active site. requires GLu 35 protonates the GLucNAc =
OH group of sugar departs
proton from Glu 35 and oxygen from linker
there is a covalent intermediate form between Asp52 and carbon 1 of hexose mur2ac hexose
covalent intermediate is hydrolysed
glu35 acts as a general base catalyst to faciitate another Sn2 attack where water displacing Asp 52 and generates product
enzyme reestablished with a ionised asp 32 protonated glu35 promted by their environments in the enzyme
leaving it unchanged due to second Sn2
transpeptidase forms crosslinks in peptidoglycan of peptide chains of pentaglycine link sugar chains increasing strength coupled with lycine side chains
displaces 1 and d alalnine to carry out this transpeptidation reaction
penicillin interferes
bacteria die from osmotic lysis
Trans
nucleophilic attack of active site serine on transpeptidase attacking carbonyl carbon preciding terminal D-alanine
pentaglycine
reaction leads to release of D-Alanine because broken in formation of acacyl covalently linked intermediate
amino terminus of pentaglycine chain of one of other peptidoglyccin chain 2 takes partn in nucleophilic attack on cqarbonyl carbon of d-ala coupled to transpeptidase enzyme = displacement of transpeptidase enzyme + formaion of peptide bond between lysine and d-alanine enzyme
= intact linker
transpeptidase enzyme displaced and enzyme is released
penicillin mimics d-ala region
strained 4 membered beta-lactam ring and 5 membered thiazolidine ring and side chain could be amoxicillin or penicillin V or G
highly reactive towards transpeptidase enzyme
serine residue on transpeptidase attacks beta lactam ring on penicillin opening it up inactivating the transpeptidase (stably derivatizes)
d-ala d-ala wedged into active site
penicillin resistance
due to overuse
beta lactamase evolved by the bacterium catalyses hydrolysis of the beta lactam ring
similar action to that of transcriptidase but its able to hydrolyse the beta lactam Adduct
clavulanic acid
beta lactamase inhibitor
membered ring breaks down upon attack of betas lactamase
product is reactive towards nucleophiles in active site off beta lactamase
= irreversible acylation of the active site Serine
