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Methods for Studying Enzymatic Reactions 71
− O
O
P
O −
O
P
O −
O
O
P
O −
OAd
− O
O
P
O −
−
O O
P O P
O − O −
OAd
O O − O O
ENZ
+
H 3 N
CO 2
−
+
H 3 N
CO 2
−
O
P
O − − O
P
O −
− O P O
OAd
− O
O
P
O −
−
P
−
O
O
P
O −
OAd
O O − O O
+
H 3 N
CO 2
−
+
H 3 N
CO 2
−
Figure 4.23 Positional isotope exchange.
position but also in the b-phosphate oxygens. Not only can such isotope
exchanges be observed, the rate of isotope exchange can be measured and
compared with the rate of the overall enzymatic reaction.
The Wnal proof that a certain species is a true intermediate in an enzymatic
reaction is by preparing the intermediate by independent chemical synthesis.
There are two criteria that must be satisWed for a potential intermediate. The
Wrst is chemical competence: the intermediate must be converted by the enzyme
to the product of the reaction (and also be a substrate for the reverse reaction if
the enzymatic reaction is reversible). The second is kinetic competence: the rate
of conversion to products must be at least as fast as the rate of the overall
enzymatic reaction.
4.6 Analysis of transition states in enzymatic reactions
Enzymatic reactions are frequently multi-step reactions. In such reactions
the overall rate of the enzymatic reaction is governed by the step having the
highest transition state energy – the rate-determining step. If we want to
determine a detailed kinetic proWle for an enzymatic reaction, it is important
to know which is the rate-determining step. Once again we can use isotopic
substitution, this time to study the existence of kinetic isotope eVects.