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  Section: General Biochemistry » Enzyme Mechanisms
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Phosphate Transfer

Enzymes that catalyze the transfer of a phosphoryl moiety between two substrates have provided excellent examples of the use of isotopes in kinetic and stereochemical studies. The enzyme hexokinase, which promotes the conversion of glucose plus ATP to glucose-6-phosphate and ADP has been the subject of kinetic studies that suggested an ordered kinetic sequence with glucose being the first substrate to add and glucose-6-P the last product to be released. Specific information on the identity of rate-limiting steps and the steady-state levels of reaction intermediates was obtained by isotope trapping studies. In its simplest form, enzyme and isotopically labeled substrate (S*) are incubated (the pulse) and rapidly diluted into excess unlabeled substrate (the chase), and allowed to react for a chosen time. Then the reaction is stopped by a quenching reagent that jumps the pH or denatures the enzyme. From the amount of E · S* converted to product versus that lost to dissociation (replacement by S gives nonlabeled product) the dissociation rate of S* from E and other ES complexes can be calculated.

This method has been used in the study of the partitioning of ES complexes in the steady state. In the case of hexokinase, the question was the partitioning of the functional E · glucose · ATP complex between product formation and substrate release. For glucose the relevant scheme is:
  E + Glc* koffGlc* E . Glc* koffATP   kc E . Glc*-6-P . ADP koffADP E · Glc*-6-P + ADP → E.
  E . Glc* . ATP

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
phosphate intermediate.

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
used to detect an isotopic exchange brought about by glutamyl phosphate formation. The synthesis of γ -18O4P-ATP containing an 18O label in the βγ bridging oxygen provides the necessary probe for finding this intermediate by means of the process below. In these experiments, isotopes are used as labels so that the fate of a particular atom may be followed throughout the course of the reaction. The appearance of 18O in the nonbridging oxygens of the β-phosphate can be measured by mass spectrometric and NMR methods. The extent of equilibration is partially inhibited by the presence of ammonia as required if glutamyl phosphate is a reaction intermediate.

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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