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Carboxytransphosphorylase

VIII. LIGAND-MEDIATED INTERACTION OF SUBUNITS AS A POSSIBLE CONTROL MECHANISM IN PROPIONIBACTERIA

From the ¹ From the Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

Phosphoenolpyruvate carboxytransphosphorylase from Propionibacterium shermanii catalyzes the conversion of P-enolpyruvate, orthophosphate, and CO₂ to oxalacetate and pyrophosphate. The enzyme occurs in three enzymatically active forms, tetramer, dimer, and monomer, but only the tetrameric and monomeric forms have been isolated from the bacteria. The dimeric form was initially observed by Haberland et al. (Haberland, M. E., Willard, J. M., and Wood, H. G. (1972) Biochemistry 11, 712–722) in reacting enzyme sedimentation centrifugation, and it was shown that dissociation of the tetrameric enzyme and association of monomeric enzyme to dimeric forms occurred during catalysis of the forward reaction. We have now shown that the association-dissociation is not caused by the substrates but by the product, oxalacetate. When the forward reaction is conducted in the presence of hydrogen peroxide, which oxidizes oxalacetate to malonate, no dimer formation is observed. In addition to oxalacetate, malate and fumarate induce the subunit interactions.

Our results show that the dimeric species is less active than the tetrameric species. Preincubation of the tetrameric enzyme at dilute concentrations in malate buffer results in a loss of 50% of the activity within 5 min. This loss in activity is strongly dependent on the concentration of protein, there being less loss of activity with higher concentrations of protein in the malate buffer. The tetramer-dimer interaction is in equilibrium since only a single peak is observed on centrifugation in malate buffer, and as the protein concentration of the tetrameric form is decreased the s_{20, obs} value decreases until it approaches that of the dimer. At the same time, there is a loss in enzymatic activity which correlates with the increase in the dimeric form.

The monomeric species has a specific activity about one-fourth that of the tetrameric species, and the activity is not affected by the presence of malate although there is total conversion of monomeric into dimeric species. The continued presence of an effector is required for maintenance of the dimeric species from either tetramer or monomer.

Sedimentation equilibrium studies in malate buffer gave a molecular weight of 224,000 for the dimeric species formed from monomeric enzyme. When tetrameric enzyme was employed a nonlinear log Y versus r² plot was observed reflecting the equilibrium between tetramers and dimers induced by the presence of an effector.

The malate-treated tetrameric enzyme is more susceptible to proteolytic attack by trypsin and chymotrypsin than the untreated enzyme.

It is proposed that the ligand-induced subunit interactions play a role in the control of the propionic acid fermentation.

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