Isotopic exchange sequential mechanism

In the presence of activator, pyruvate, the substrate saturation curves of the R. ruhrum ADP-Glc PPase are hyperbolic at low temperatures. Using kinetic studies its reaction mechanism was studied. The product inhibition patterns eliminated all known sequential mechanisms except the ordered BiBi or Theorell—Chance mechanisms. Small intercept effects suggested the existence of significant concentrations of central transis-tory complexes. Kinetic constants obtained in the study also favored the ordered BiBi mechanism. In addition studies using ATP-[ P]-pyrophosphate isotope exchange at equilibrium supported a sequential-ordered mechanism, which indicated that ATP is the first substrate to bind and that ADP-Glc is the last product to... 

With respect to mechanism of action, the most extensive kinetic and equilibrium exchange studies have been carried out on monofunctional 10-formyl-H4-folate synthetase from Cl. cylindrosporum [84]. The data support a random sequential mechanism that does not involve the formation of freely dissociable intermediates. The most likely mechanism, however, is not concerted but probably involves the formation of a formyl phosphate intermediate, since the synthetase catalyzes phosphate transfer from carbamyl phosphate but not acetyl phosphate to ADP with H 4-folate serving as an activator. Carbamyl phosphate is an inhibitor of 10-formyl-H 4-folate synthesis - an inhibition that can be eliminated only when both ATP and formate are present in accord with the concept that it spans both sites [85]. It would be of considerable interest to attempt to demonstrate positional isotope exchange employing [, y- 0]ATP for this enzyme in order to further implicate an enzyme-bound formyl phosphate species [86]. 

ISOTOPIC EXCHANGE AT CHEMICAL EQUILIBRIUM IN SEQUENTIAL MECHANISMS... 

Most isotopic exchange experiments where the mechanism is sequential are conducted under equilibrium conditions, and most of the formal treatments have dealt with the equilibrium (Boyer, 1959 Morales et al, 1962 Boyer Silverstein, 1963, 1964 Allison et al, WIl Puiich AUison, 1980). However, some treatments have dealt with isotopic exchange in the steady state (Alberty et al, 1962 Cleland, 1967 Boyer, 1978 Purich Allison, 2000). 

Table 2. Isotope exchange-rate expressions for sequential mechanisms CPurich Allison, 1980, 2000). 

Table 2 summarizes the isotope exchange rate expressions for selected common sequential mechanisms, in addition to the Ordered Bi Bi mechanism outlined above. Since there is no such mechanism as Rapid Equilibrium Random Bi Bi without a dead-end complex, at least a dead-end complex EBQ must form (mechanism 4). If both dead-end complexes, EBQ and EAP, are formed, an extra KrP/KspB term is added in the denominator of rate equation for mechanism 4 K p represents the dissociation constant of P from the EAP complex. 

Nevertheless, isotopic exchange under nonequilibrium conditions were employed to establish the order of product release or the order of substrate addition in sequential mechanisms. 

They postulated that either end of the rc-allyl species, which forms initially, has an equal probability of collapsing on a lattice oxygen, and the exchange observed with allyl alcohol indicates that this process is rapid and reversible. The same isotopic product distribution is expected as for the sequential hydrogen removal mechanism shown in Scheme 5.1 and the presence of a a C-O bond in -O-CH2-CH-CH2 could reduce the activation energy for removal of the second hydrogen. 

---