Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

278 Appendix 4
(2) Intramolecular acid catalysis (carboxylic acid will be protonated at pH 4).
(3) (a) Phenoxide ion attacks to form Wve-membered ring. EVective concentration
7:3 10 4 m. Large rate acceleration due to intramolecular
nucleophilic attack, Wve-membered ring.
(b) Either (i) general base-catalysed attack of water or (ii) nucleophilic
attack by active site aspartate to give covalent ester intermediate (similar
to haloalkane dehalogenase). To examine mechanism (ii) could try
to detect covalent intermediate (e.g. by stopped Xow methods). Rate
enhancement through transition state stabilisation, bifunctional catalysis,
etc.
(4) In the Wrst catalytic cycle, the oxygen atom introduced into the product
comes from the aspartate nucleophile, via hydrolysis of the ester intermediate.
In subsequent cycles 18 O label becomes incorporated into the active site
aspartate and is transferred to product.
Chapter 4
(1) 787 units mg 1 28 mg mmol 1 ¼ 28 mmol product min 1 mmol 1 enzyme.
So k cat ¼ 0:47 s 1 .
(2) Use Lineweaver–Burk or Eadie–Hofstee plot. v max ¼ 6:0 nmol min 1 ,
K M ¼ 0:71m m: k cat ¼ 2:0s 1 ,k cat =K M ¼ 2, 800 m 1 s 1 .
(4) Retention of stereochemistry. Not consistent with an S N 2-type
displacement.
(5) Imine linkage formed between aldehyde group and e-amino group of
an active site lysine residue. Enzymatic reaction goes with retention of
conWguration at phosphorus, whereas non-enzymatic reaction goes with
inversion. Suggests that non-enzymatic reaction is a single displacement,
whereas enzymatic reaction is probably a double displacement reaction
proceeding via a phosphoenzyme intermediate.
(6) Could try to detect phosphoenzyme intermediate using 32 P-labelled substrate.
In D 2 O should see 2 H incorporation into acetaldehyde.
Chapter 5
(1) Hydrolysis of acyl enzyme intermediate is rate-limiting step in this case.
Rapid formation of acyl enzyme intermediate, releasing a stoichiometric
amount of p-nitrophenol, followed by a slower hydrolysis step.
(2) Mechanism as for chymotrypsin, via acyl enzyme intermediate. Phosphorylation
of active site serine by organophosphorus inhibitors gives a tetrahedral
adduct resembling the tetrahedral intermediate in the mechanism,
which is hydrolysed very slowly. DiVerences in toxicity due to: (i) presence
of sulphur on parathion, which de-activates the phosphate ester (in insects
this is rapidly oxidised to the phosphate ester, which then kills the