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Enzymatic Addition/Elimination Reactions 207
Problems
(1) In the 3-dehydroquinate dehydratase reaction, how would you identify the
active site lysine residue which forms an imine linkage with the substrate
(2) The following intramolecular addition reaction is catalysed by a cycloisomerase
enzyme from Pseudomonas putida. When the cycloisomerase
reaction was carried out in 3 H 2 O, product was found to contain tritium
at C-5 and was found to have the 4S, 5R stereochemistry. Deduce whether
the addition reaction occurs with syn- or anti- stereochemistry, and suggest
a mechanism for the reaction.
− O 2 C
CO 2
−
muconate
cycloisomerase
− O 2 C
5
4
O
O
(3) Dihydroxy acid dehydratase is an iron–sulphur cluster-containing hydratase
enzyme which catalyses the interconversion of ab-dihydroxy acids with
a-keto acids. Suggest possible mechanisms for this enzyme, and predict
what would be observed for each mechanism if an a- 2 H dihydroxy acid
substrate was incubated with the enzyme.
HO
R
OH
CO 2
−
dihydroxy acid
dehydratase
[FeS]
R
O
CO 2
−
+ H 2 O
(4) S-Adenosyl homocysteine hydrolase catalyses the conversion of S-adenosyl
homocysteine to adenosine and homocysteine. Given the following data,
suggest a mechanism for this enzyme: (i) the enzyme contains a catalytic
amount (1 mole per mole enzyme sub-unit) of tightly bound NAD þ (ii) the
enzyme catalyses the exchange of the C-4 0 hydrogen (H ) with 2 H 2 O.
+
H 3 N
N
S
N
O
*H
CO −
2 HO OH
S-adenosylhomocysteine
NH 2
N
N
+H 2 O
S-adenosylhomocysteine
HO
hydrolase
SH
NAD + + +
H 3 N
CO −
2
N
N
O
HO OH
NH 2
N
N