Chymotrypsin reactions Flashcards
explain peptide hydrolysis by H2O without catalyst
- H2O acts as a nucleophile, lone pair donates to electron deficient C
- neutral O is NOT a good nucleophile
- tends to hold onto its own electrons
- make unfavourable O+ transition state
- C maintains 8 valence electrons by allowing upper O to take back a bond
- this leads to OXYANION transition state (Note C is not sp3 tetrahedral)
- transition state may break down with N as leaving group
- oxyanion O returns to bond C
- Carboxylate C must give up a bond to maintain 8 valence electrons
- if Ntakes back excess, the peptide bon breaks
How can the transition state break down by reuturning electrons to O?
- carboxylate C may give up the excess bonding electrons to original nucleophilic O
- the C-O bond breaks
- reactants are back to the starting point, NO NET RXN
How is chymotrypsin better at breaking bonds?
- done in two steps
1) a nucleophilic group -X: in the enzyme attacks the peptide C=O to split off the C-terminal half of the substrate, but leave the N-terminal covalently bonded to the enzyme group-X (acyl- enzyme intermediate)
2) brings in H2) to release the N-terminal half, and restores the enzyme group-X: to its original state
What does acyl refer to
acid
What is the catalytic triad
- 40 reactions per second for 1 chymotrpsin; 1 reaction in 10 years for H2O
- chymotripsin uses a better nucleophile in form on the CATALYTIC TRIAD
- three amino aicds that line up side by side in correctly folded chymotrypsin and cooperate for maximum effectiveness
- ASP 102: Neg charge favorus a positive charged partner
- His 57 would be positive if it could capture H+
- pKa near 7, gets H+ from Ser
- Ser 195 could give up H+ if it shares a lone pair with a suitable atom
- Combined effect makes Ser 196 into better nuclephile
- transition state is stablized by the oxyanion hole
What do numbers indicate?
- posiiton in polypeptide sequence (asp 102, His 57)
Explain step 1 of the catalytic triad
- His 57 (acts like a general base) removes H+ at the moment of reaction to help Ser
- negative charge of Asp helps His act like a base
- the oxyanion hole helps pull yhe O- into transition state by H-bonding to backbone NH groups of Ser and Gly 193
- H-bonds are amined at location matching a tetrahedral C
*SER- O initiates attach, His acts as general base removing H+ from ser
* OXYANION HOLE helps substrate reach first tranition state
What is the formaltion of the first transition state
- oxyanion hole pulls O- into transition state
- complementary to transition state, favours tetrahedral carboxyanion
*note: carbon is never leaving group
*note his acting as general acid
What is the breakdown of the 1st transition state FOrmation of Acyl- Enzyme Intermediate
- NH group of substrate acts as leaving group
- His 57 acts as a general acid, donating H+ to leaving group
- C-term peptide leaves
- N-terminal peptide remains covalently bound: the Acyl enzyme intermediate
Explain step 2: nucleophilic attack
- water now enters the catalytic site
- His-57 acts as a general base removing H+ from water
- water becomes a better nucleophile attacks the acyl-enzyme C=O
What is the formation of the second transition state
- oxyanion hole stabilizes the transition state configuration
- green is the leaving group
Explain the breakdown of second transition state formation of products0 his 56 acts
WHhat role does His-57 play in the catalytic mechanism of chymotrypsin?
Acts as a general acid or base
- make sure you can explain why
How do we know the chymotrypsin catalytic cycle is complete
- chymotrypsin is back to its original state: reaction cycle can repeat 40 times per second
- the mechanism was determined by structural studies
- replace Asp with Ala: 10% normal rate
- replace His with Lys: 0.1% normal rate (lys is a good base but poor acid)
- replace ser with ala (little catalytic reaction
- catalytic mechanism or trypsin and elastase is identical
What is enzyme assay and detection
- enzymes speed up reaction rate in protortion to amount of enzyme present
- enzyme assay is the process of measuring enzyme-catalyzed reaction rate
- enzyme kinetics is mathemetical analysis of how rate varies as a function of substrate oncentration: kinetics can be used to test reaction mechanism
- measure rates: rate of dissappearence of reactant or apprearance of product
- Equation format: enzyme name placed above (bc not consumed)
- measure volume of O2 released
- Measure rise in pH as [H+] decreases
How are artificial substrates used for analysis of trypsin
- direct analysis of trypsin reaction products is too time consuming
- artificial substrate is used (in ex is peptidyl nitroanilide, not the lys and trp will hydrolyse that bond)
- an atrificial subtrate it a “molecular look alike” for the real substrate
- rxn product is distinctly coloured (easy to measure)
- trypsin recognizes lys and the peptide chain upstream of the Lys (peptide after lys less important)
- trypsin accepts any primary amino group in position after lys or arg but not proline (secondary amino group)
What is shown in natural substrates after conversion to product
- UV absorbance change
- ex: lactate dehydrogenase uses 2H atoms from reduced form of nicotinamide adenine dinucleotide (NADH) and transfers them to pyruvate lactate
- his uses up NADH, which absorbed ultraviolet light at 340 nm, whereas the NAD+ product does nto absorb
- overall absorbance decreases as reaction proceeds
(DW about the rxn just udnerstand process and used to measure amount of substrate/process)
What are chromophores
- parts of molecules with conjugated double bond ex: N-C=C-C=C (alternating single double) or aromatic rings, which absorb UV light
- coloured compounds absorb between 400-700
- natural biochemical chromophores frequently absorb in the UV range 200-400
- larger chemicals absorb at longer wavelengths
How is absorbance measured
- absorbance is measured with a spectrophotometer
- mean has intensity Io (after light absorbed intensity I)
- absorbance A= log(Io/I)
- absorbance is a measure of concentration
A= cle (l is in cm)
