1 enzymes

what type of catalysis is used by enzymes and why

• dont use specific because [H₃O⁺] and [HO^-] are very low at physiological pH (approx 7_

• but general is not a problem

how can enzymes act as general catalysts

bring reactants together via non-covalent interactions and can place catalytic functional groups in the right location to enable catalysis to occur

show the steps of an enzyme-catalysed reaction

what makes RNA susceptible to hydrolysis

the presence of the 2’-OH group adjacent to the phosphate - the chain can be cleaved to form a cyclic product

structure of RNA

show how RNA can be hydrolysed by bases

why is DNA not hydrolysed in the same way as RNA

it lacks the OH groups needed - the base would instead have to attack directly at the P

how is an RNase reaction catalysed

pKa?

catalysed by two histidines, pKa ~ 7

one is deprotonated and one is protonated at neutral pH

show deprotonated histidine

show protonated histidine

show the two histidines involved in the RNase reaction

what do they act as in the reaction?

show the mechanism of RNase

next step of RNase mechanism

next step of RNase

product of this stage of RNase mechanism

what is the RNase mechanism dependent on

dependent upon one histidine being protonated and one deprotonated

when is maximum activity for RNase and why

• maximum activity at pH 6

• below pH 6 both histidines are protonated

  • above pH 6 both histidines are deprotonated

what change may be made to the RNase reaction and what is the effect of this

changing either of His12 or His119 to alanine dramatically reduces the rate of reaction

show Ala12/Ala19

how can this be made to work with Ala119 instead

nitrophenolate anion is a good leaving group - doesn’t need to be protonated by His119

show the resonance forms of the nitrophenolate ion and explain why is is a good leaving group

why does nature want to cleave peptide/amide bonds

RNA is inherently unstable

in contrast, an amide bond is essentially indefinitely stable in physiological conditions

how can the hydrolysis of an amide bond be performed

using enzymes called proteases

where are the important roles of proteases (4)

digestion, cell signalling, blood clotting, inflammation

type(s) of catalyst used by serine protease

acid/base and nucleophilic

what are the 3 residues that serine protease relies on

what is this called?

Ser

His

Asp

Catalytic triad

pKa of serine

approx 14

show serine protease’s catalytic triad and how it links to the backbone

show the first step of serine protease’s mechanism with a peptide bond

describe the roles of the groups involved

what is the product of this step of serine protease’s mechanism

explain any charges and relevent intermolecular forces

His protonated - general acid

Asp helps stabilise His positive charge

tetrahedral intermediate - active site has H bonding to help stabilise negative charge

what is the next stage in the serine protease mechanism

comment on the leaving group?

NH not good leaving group but protonate with His as it leaves

product of this stage of the serine protease mechanism

what happens next in the serine protease mechanism

what is the product of this stage of serine protease

what happens next in serine protease mechanism

product of this stage of serine protease mechanism

enzyme in original state, product leaves

how is it clear this is the reaction mechanism?

if any of the three amino acids of the enzyme are replaced, there is a large drop off in enzyme activity

shows it is essential

what makes enzyme activity pH-dependent

necessity of charges (or lack of charges) on certain amino acid side chains

pKa of Asp

4

pKa of His

7

pKa of Ser

14

what are the three serine proteases that are chiefly responsible for digesting dietary protein in small intestines

trypsin

chymotrypsin

elastase

how do trypsin, chymotrypsin and elastase cleave

cleave amide bonds adjacent to specific amino acid residues

where does the selectivity of trypsin, chymotrypsin and elastase come from?

a binding pocket next to the active site

label this to describe the residues on either side of the cleavage site as well as where the binding pockets are

what does this protease bind?

large, aromatic side chains

what does this protease bind

positively-charged side chains

what does this protease bind

small side chains