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The mechanism of the phosphoglucomutase reaction. Studies on rabbit muscle phosphoglucomutase with flux techniques.

ABSTRACT: 1. The kinetics of phosphoglucomutases from different sources are discussed and it is concluded that on the available evidence there are in all cases three possible mechanisms for the reaction. These are an indirect transfer of phosphate involving the phosphoenzyme (mechanism 1), a direct transfer of phosphate (mechanism 2), and an intermolecular transfer of phosphate from glucose 1,6-diphosphate to the substrate (mechanism 3). Conventional net flux measurements are shown not to differentiate between these mechanisms. 2. Flux equations are developed and it is shown that there are three flux ratios that characterize and distinguish between the mechanisms. 3. To examine these flux ratios induced-transport tests are described with (14)C- and (32)P-labelled substrates. The fluxes determined with (14)C- and (32)P-labelled substrates are also compared at chemical equilibrium. 4. With rabbit muscle phosphoglucomutase the results of these tests were completely consistent with mechanism 1 and unequivocally excluded any substantial part of the reaction proceeding by mechanism 2 or mechanism 3. Evidence was also obtained for an isomerization of the phosphoenzyme with an apparent rate constant about 4.5x10(7)sec.(-1). Taking into account the activity coefficients of the substrates the true rate constant appears to be about one-sixth of this value. 5. Isotope effects and non-ideal behaviour of the solutions are discussed and the activity coefficients of the substrates are shown to be equal by measurement of the depression of freezing point. It is concluded that these factors do not influence the tests significantly. 6. Alternative mechanisms are considered and it is concluded that the tests show that the glucose residue is transferred directly, that the phosphate is transferred indirectly with one intermediate phosphate, and that there is an isomerization of the free phosphoenzyme without reference to any other details of the reaction. Further, no assumptions are required about the constancy of rate constants. 7. The relative merits of induced transport and product inhibition for detecting isomerization of the enzyme are discussed. It is concluded that the induced-transport test is more sensitive and that its interpretation is less equivocal. 8. The application of the tests to other enzyme systems is briefly considered.

SUBMITTER: Britton HG

PROVIDER: S-EPMC1187194 | biostudies-other | 1968 Nov

REPOSITORIES: biostudies-other

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Project description:The mechanism of rabbit muscle phosphofructokinase was investigated by measurement of fluxes, isotope trapping and steady-state velocities at pH8 in triethanolamine/HCl buffer with 4 mM free Mg2+. Most observations were made at I0.2. The ratio Flux of fructose 1,6-bisphosphate----fructose 6-phosphate/Flux of fructose 1,6-bisphosphate----ATP at zero ATP concentration increased hyperbolically from unity to about 3.2 as the concentration of fructose 6-phosphate was increased. Similarly, the ratio Flux of fructose 1,6-bisphosphate----ATP/Flux of fructose 1,6-bisphosphate----fructose 6-phosphate at zero fructose 6-phosphate concentration increased from unity to about 1.4 as the concentration of ATP was increased. The addition of substrates must therefore be random, whatever the other aspects of the reaction. Further, from the plateau values of the ratios, it follows that the substrates dissociate very infrequently from the ternary complex and that at a low substrate concentration 72% of the reaction follows the pathway in which ATP adds first to the enzyme. Isotope-trapping studies with [32P]ATP confirmed that ATP can bind first to the enzyme in rate-limiting step and that dissociation of ATP from the ternary complex is slow in relation to the forward reaction. No isotope trapping of [U-14C]-fructose 6-phosphate could be demonstrated. The ratios Flux of ATP----fructose 1,6-bisphosphate/Flux of ATP----ADP measured at zero ADP concentration and the reciprocal of the ratio measured at zero fructose 1,6-bisphosphate concentration did not differ significantly from unity. Calculated values for these ratios based on the kinetics of the reverse reaction and assuming ordered dissociations of products or a ping-pong mechanism gave values very significantly greater than unity. These findings exclude an ordered dissociation or a substantial contribution from a ping-pong mechanism, and it is concluded that the reaction is sequential and that dissociation of products is random. Rate constants were calculated for the steps in the enzyme reaction. The results indicate a considerable degree of co-operativity in the binding between the two substrates. The observations on phosphofructokinase are discussed in relation to methods of measurement and interpretation of flux ratios and in relation to the mechanism of other kinase enzymes.

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The mechanism of the phosphoglucomutase reaction. Studies on rabbit muscle phosphoglucomutase with flux techniques.

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