Rabbit muscle phosphofructokinase. II. Product and dead end inhibition

J. Bar Tana, W. W. Cleland

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Abstract

Product inhibition patterns and patterns for dead end inhibition by 1 deoxy D fructose 6 P and the chromium complex of ATP have been determined for rabbit muscle phosphofructokinase at pH 7.4, 25°. Nucleotides are mutually competitive, as are sugars, with nucleotides being noncompetitive versus sugars and vice versa, except that MgATP appears competitive versus fructose P2. These data establish that the kinetic mechanism is random, with the formation of a dead end E MgADP fructose 6 P complex, but little or no E MgATP fructose P2 complex. Quantitative analysis of the inhibitions and comparison with the initial velocity data from previous studies suggest that the rapid equilibrium assumption holds in the reverse reaction, but that with MgATP and fructose 6 P as substrates the rate constants for release of these substrates are less than Vmax, with the result that the initial velocity patterns appear parallel or nearly so. In the absence of nucleotides, fructose 6 P binds to phosphofructokinase 30 fold more tightly than 1 deoxyfructose 6 P, showing that the 1 OH is important for binding, but in the presence of MgATP the affinities are the same, suggesting that the conformation change which normally produces catalysis by generating compression between the 2 substrates causes deoxyfructose 6 P and MgATP to move closer to each other and thus be more tightly bound when the 1 OH is missing. Similarly, in the absence of sugars MgATP is bound 20 fold more tightly than the chromium complex of ATP, but in the presence of fructose 6 P the affinities are the same. The chromium complex of ADP does not show the effect, although its affinity for free enzyme is the same as that of the chromium complex of ATP.

Original languageEnglish
Pages (from-to)1271-1276
Number of pages6
JournalJournal of Biological Chemistry
Volume249
Issue number4
StatePublished - 1974

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