Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

282 Appendix 4
Chapter 8
(1) Treat with substrate and NaB 3 H 4 (or 14 C-substrate þ NaBH 4 ), degrade
labelled enzyme by protease digestion, purify labelled peptide and sequence.
(2) Overall syn-addition. Attack of carboxylate onto ab-unsaturated carboxylic
acid to give enolate intermediate, which protonates on same face.
(3) Possibilities are: (i) elimination of water to give enol intermediate, which
protonates in b-position; (ii) 1,2-hydride shift from a- tob-position (cf.
Pinacol re-arrangement). In mechanism (ii) an a- 2 H substituent would be
transferred to b-position, in mechanism (i) probably not (unless single base
responsible for proton transfer). No intramolecular proton transfer
observed in practice, so probably mechanism (i).
(4) Oxidation of C-3 0 hydroxyl group by enzyme-bound NAD þ . Formation of
C-3 0 ketone assists elimination of methionine by E1cb mechanism, giving
ab-unsaturated ketone intermediate. Addition of water at C-5 0 , followed by
reduction of C-3 0 ketone, regenerating enzyme-bound NAD þ .
(5) Isochorismate synthase could be 1,5-addition of water, but all three reactions
can be rationalised by attack of an enzyme active site nucleophile at C-2
and allylic displacement of water, giving a covalent intermediate. Attack
of water at C-6 gives isochorismate. Attack of ammonia at C-6 or C-4,
followed by elimination of pyruvate, gives anthranilate or p-aminobenzoate,
respectively.
EnzX −
CO 2
−
EnzX
CO 2
−
OH 2 or NH 3
OH
H +
O
CO 2
−
NH 3
O
CO 2
−
Chapter 9
(1) Use of PLP as a four-electron sink. Formation of threonine-PLP adduct,
followed by deprotonation in a-position, gives ketimine intermediate.
Deprotonation at b-position possible using imine as electron sink. Either
one-base or two-base mechanisms possible for epimerisation process.
(2) Formation of aldimine adduct followed by a-deprotonation gives abunsaturated
ketimine intermediate. This can be attacked by an active site
nucleophile at g-position to covalently modify enzyme.
(3) Formation of aldimine adduct with inhibitor followed by a-deprotonation
gives a ketimine intermediate which is a tautomeric form of an aromatic
amine. Rapid aromatisation gives a covalently modiWed PLP adduct.