Abstract
Rabbit muscle pyruvate kinase detritiates phosphoenolpyruvate 3-tritium under conditions of the net forward reaction prior to the release of pyruvate. This exchange requires that release of pyruvate is not rapid with respect to reversal of the proton transfer steps involved in the catalysis of its enolization. This conclusion is consistent with the low tritium isotope effect that is observed for the hydrogen exchange on pyruvate that is activated by ATP. An intrinsic isotope effect for the proton abstraction step as high as 26 can be expected since such high values were observed in the enzymatic enolization of pyruvate activated by other phosphate compounds. Considerable exchange of tritium from tritiated water into remaining phosphoenolpyruvate during the course of the forward pyruvate kinase reaction is observed, indicating that the enzyme-bound pyruvate-ATP complex that is generated in the forward reaction can return to substrate form at a significant rate relative to product release. A kinetic analysis indicates that the release of phosphoenolpyruvate and ADP may be a rate-determining factor in this exchange rate. Thus, the conclusions of earlier workers that muscle pyruvate kinase follows rapid equilibrium kinetics require re-examination. The effects of varying pH, mono- and divalent cations, temperature, and alternative activators on the rates of proton exchange are reported.
Highlights
Previous experiments by Harrison et al [14], in which the possiblility of a phosphoryl-enzyme intermediate was eliminated using the test of pyruvate to PEP exchange is more demanding of the mechanism than the tritium exchange test since the latter does not require that free pyruvate be formed from PEP in the absence of XDP
The rates of loss of the proton signal of pyruvate and of the appearance of tritium in water were compared with either ATP or an ATP analog to activate the exchange
The discrimination against tritium incordetermining for the forward reaction, Eassshould equal Eaeq poration into pyruvate has previously been studied at pH 7 by since the preliminary equilibrium of all enzyme complexes Simon et al [20] with a discrimination factor of about 6, as should partition the enzyme in the same way as under the equi- found here at pH 7.3 with Mg2f
Summary
Rabbit muscle pyruvate kinase detritiates phosphoenolpyruvate 3-tritium under conditions of the net forward reaction prior to the release of pyruvate This exchange requires that release of pyruvate is not rapid with respect to reversal of the proton transfer steps involved in the catalysis of its enolization. This conclusion is consistent with the low tritium isotope effect that is observed for the hydrogen exchange on pyruvate that is activated by ATP. The over-all reaction catalyzed by pyruvate kinase consists in the transfer of the phosphoryl group of phosphoenolpyruvate to Al)l’ and the transfer of a proton from water to form the methyl group of pyruvate This dual functionality is illustrated by the following proposed reaction sequence in which enolpyruvate is considered to be an enzyme-bound intermediate. Rose on steady state kinetics, is not valid and that the rate-determining step is the release of products
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