In this case the reaction is allowed to reach steady-state turnover, and the solution is either stopped by quench or chased by addition of excess unlabeled substrate followed by a delay sufficient for several turnovers then addition of quench. The presence of a difference in the level of the labeled product obtained by the two procedures represents the concentration of E · Glc · ATP* complex in the steady state, which is approximately 50% of ET, the total enzyme concentration. The observed steady-state and pretransient rates are consistent with steps kc and k−c being at equilibrium relative to koffADP, which is typical for many phosphotransfer enzymes in which the chemical steps are generally not rate limiting. Additional information can be obtained by using the label in the second substrate (i.e., [γ -32P]ATP) and following a similar protocol, which thereby allows calculation of the dissociation rate of ATP from E · Glc · ATP. In this case E · Glc · ATP* is approximately 25% of ET, which requires that koffATP compete with the dissociation of ADP (koffADP ) from E · Glc-6-P · ADP. In this manner the individual rate constants for hexokinase were largely determined and the order of substrate association was verified.
Isotopic labeling has also been cleverly used to demonstrate the existence of enzyme-bound intermediates that do not readily dissociate into solution. The enzyme glutamine synthetase catalyzes the formation of glutamine from ATP and ammonia possibly through a tightly bound glutamyl
If the formation of glutamyl phosphate were reversible and occurred in the absence of ammonia, then the presence of a symmetric torsion motion at the cleavage site might be
Isotopic labeling studies of phosphotranferase reactions culminated in the synthesis of ATP chiral at the γ -phosphorus. Chirality was achieved by the synthesis of [γ -16O,17O,18O]ATP of one configuration, and the analysis of its chirality was achieved by stereochemically controlled transfer of the γ -phosphoryl moiety to (S)- propane-1,2-diol where the absolute configuration was determined by a chemical/mass spectrometric sequence. The observation of inversion of configuration has been accepted as evidence of an “in-line” displacement mechanism at phosphorus by the two bound substrates; the observation of retention of configuration was used to implicate the existence of a phosphoryl enzyme intermediate in the phosphoryl transfer process. For hexokinase, our case study, the finding is one of inversion, consistent with a direct transfer mechanism.
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