Positional isotope exchange

Selected entries from Methods in Enzymology [vol, page(s)] Equilibrium isotope exchange study of kinetic mechanism, 249, 466 site-directed mutagenesis of Escherichia coli enzyme, 249, 93 positional isotope exchange studies, 249, 423 product inhibition studies of three substrates three products reactions, 249, 207-208. 

See Positional Isotope Exchange Bridge-to-Nonbridge Oxygen Exchange... 

Cleland introduced the net rate constant method to simplify the treatment of enzyme kinetic mechanisms that do not involve branched pathways. This method can be applied to obtain rate laws for isotope exchange, isotope partitioning, and positional isotope exchange. Since the net-rate constant method allows one to obtain VraaJKra and in terms of the individual rate constants, this method has greatest value for the characterization of isotope effects on and Kj. Because only... 

Positional isotope exchange ( PIX ) is a very valuable technique in determining enzyme mechanisms, particularly those utilizing ATP, GTP, or another NTP substrate -. For example, the nucleotide substrate can be labeled with in bridging (i.e., P—O—P or phos-phoanhydride oxygen) and/or its nonbridging positions. 

POSITIONAL ISOTOPE EXCHANGE BRIDGE-TO-NONBRIDGE OXYGEN EXCHANGE... 

When 31P is bonded to lsO the chemical shift of the 31P is altered by 0.0206 ppm from that when the phosphorus is bonded to leO. This allows lsO labels introduced into phospho groups to serve as tracers which can be followed continuously during reactions.683 The technique is useful in studies of stereochemistry (see Section 2) and for examination of positional isotope exchange.690 This latter technique is often used with ATP containing lsO in the P,y-bridge position. If an enzyme transfers the terminal (y) phospho group to an acceptor via a phosphoenzyme but without loss of the ADP, we may expect positional isomerization. The lsO will move between the P,y-bridge position and... 

Figure 12-21 The 31P NMR spectrum at 101.2 MHz of Pa of isotopically labeled ADR This was recovered from an experiment in which ADP containing 87 atom % of lsO in all four oxygens around Pp was allowed to undergo partial ( 20%) nonenzymatic hydrolysis to AMP and P . Peaks 1 represent the species containing no lsO bonded to Pa. Peaks 2 represent all species with lsO in the Pa-0-Pp bridge, and peaks 3 represent species with lsO in nonbridging positions at pa. These have undergone positional isotope exchange. From Lowe and Tuck.691...

Positional isotope exchange experiments conducted on creatine kinase,45 hex-okinase,46 and pyruvate kinase47 have provided no evidence in favor of a dissociative mechanism. In each case, the data are consistent with a straightforward nucleophilic attack on phosphorus to generate a trigonal bipyramidal intermediate.47... 

Recently Midelfort and Rose (4) introduced a positional isotope exchange technique that is also suitable for study by the chemical shift technique. Briefly, this technique follows the exchange of label from one part of a substrate to another due to rotational equivalence of some intermediate. The method was first applied to glutamine synthetase in the reaction ... 

The positional Isotope exchange has also been measured with 31p-nmr In the reverse reaction of carbamyl phosphate synthetase ... 

Figure 5.60 Positional isotope exchange. The non-bridge oxygens of a phosphate are prochiral (enantiotopic if X and R are achiral, diastereotopic if they are not).The occurrence of positional exchange can be readily followed by NMR, with the P signals increasing in shielding in the order and the isotope shift increasing with bond order.

In the presence of the incompetent acceptor 4 -deoxylactose, there is no positional isotope exchange with UDPGal, nor is there any sign of enzyme inactivation by 5-fluoro-ot-galactosyl fluoride, even at high concentrations and in the presence of UDP. 

The conformations in Figure 5.65 again suggest an S i mechanism. Positional isotope exchange in apparently unreacted glucosyl phosphate would not be expected for this mechanism, unless for some reason the glycosylation of the acceptor was reversible in the ternary complex. The evidence from potato starch phosphorylase favours a double displacement mechanism there is no... 

The position of equilibrium, as would be expected, lies strongly towards the pyranose form ([/]/[p] = 0.057), " the furanose form being disfavoured by the cis interactions of hydroxyalkyl chain on C4, OH C2 and pyrophosphate on Cl. A positional isotope exchange experiment, monitored by the isotope effect... 

Figure 5.67 Reaction catalysed by UDPgalactopyranose mutase, illustrating positional isotope exchange.

To examine the kinetic competence of y-glutamyl phosphate formation at the active site of glutamine synthetase, the technique of positional isotope exchange (PIX) was introduced to studies of the mechanism of phospho transfer (85). This experiment circumvented the problem of the instability of the intermediate, which complicated direct kinetic experiments. In the PIX experiment, the rate at which glutamine synthetase catalyzes reaction (25) was measured. 

After completion of the first sequence, carboxybiotin moves to the second site, where the carboxyl group is transferred from biotin to pyruvate, forming oxal-oacetate. In essence, a proton and a carboxyl group trade places in this step. Isotope effects indicate that proton removal from pyruvate is not concerted with carboxylation (60, 61). The lack of positional isotope exchange (62) during this process presumably is because the active complex is isolated from solvent, rather than because of a lack of exchangeable sites. 

A more subtle exchange, which is indicative of internal return in the formation of an intermediate, can be detected in some enzymes through the use of what Middelfort and Rose call positional isotope exchange (32). However, this class of experiments has also given no evidence of an intermediate in that no positional isotope exchange is observed (43). Together, the exchange results show that all the reaction components, biotin, ATP, bicarbonate, and substrate, are necessary in order for an enzyme to produce A-carboxybiotin. 

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