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Methods for Studying Enzymatic Reactions 79
H 3 C
PO 3
2−
3 H
2 H
C-P lyase
H 3 C
3 H
1 H
2 H
− O 2 C
1 H
2 H
3 H
1R-ethyl phosphonate
2S acetate
(5) The enzyme phosphonoacetaldehyde hydrolase catalyses the conversion of
phosphonoacetaldehyde to phosphate and acetaldehyde, as shown below.
The enzyme requires no cofactors, but is inactivated by treatment with
phosphonoacetaldehyde and sodium borohydride. Deduce which amino
acid side chain is involved in the catalysis and suggest a possible mechanism.
H
O
O
P
O −
O −
phosphonoacetaldehyde
hydrolase
H
O
CH 3
+ PO 4
3−
[ 17 O, 18 O]-Thiophosphonoacetaldehyde was prepared with the stereochemistry
shown below, and incubated with the enzyme in H 16
2
O. The resulting
thiophosphate was analysed and found to have the S conWguration. Deduce
whether the reaction proceeds with retention of inversion of conWguration
at the phosphorus centre. Comment on the implications for the enzyme
mechanism.
The same reaction is catalysed by aniline (PhNH 2 ), but at a much slower
rate. Using the labelled substrate for the aniline-catalysed process, the
thiophosphate product was found to have the R conWguration. Explain
these observations.
H
O
S
P
17 O −
18 O −
(6) How would you attempt to obtain further evidence for the intermediate
implied in Problem 5
Further reading
Enzyme puriWcation
R.K. Scopes (1987) Protein puriWcation: Principles and Practice. Springer-Verlag, New
York.