Bacterial acetone carboxylase catalyzes the ATP-dependent carboxylation of acetone to acetoacetate with the concomitant production of AMP and two inorganic phosphates. The importance of manganese in Rhodobacter capsulatus acetone carboxylase has been established through a combination of physiological, biochemical, and spectroscopic studies. Depletion of manganese from the R. capsulatus growth medium resulted in inhibition of acetone-dependent but not malate-dependent cell growth. Under normal growth conditions (0.5 M Mn 2؉ in medium), growth with acetone as the carbon source resulted in a 4-fold increase in intracellular protein-bound manganese over malate-grown cells and the appearance of a Mn 2؉ EPR signal centered at g ‫؍‬ 2 that was absent in malate-grown cells. Acetone carboxylase purified from cells grown with 50 M Mn 2؉ had a 1.6-fold higher specific activity and 1.9-fold higher manganese content than cells grown with 0.5 M Mn 2؉ , consistently yielding a stoichiometry of 1.9 manganese/␣ 2 ␤ 2 ␥ 2 multimer, or 0.95 manganese/␣␤␥ protomer. Manganese in acetone carboxylase was tightly bound and not removed upon dialysis against various metal ion chelators. The addition of acetone to malate-grown cells grown in medium depleted of manganese resulted in the high level synthesis of acetone carboxylase (15-20% soluble protein), which, upon purification, exhibited 7% of the activity and 6% of the manganese content of the enzyme purified from acetone-grown cells. EPR analysis of purified acetone carboxylase indicates the presence of a mononuclear Mn 2؉ center, with possible spin coupling of two mononuclear sites. The addition of Mg⅐ATP or Mg⅐AMP resulted in EPR spectral changes, whereas the addition of acetone, CO 2 , inorganic phosphate, and acetoacetate did not perturb the EPR. These studies demonstrate that manganese is essential for acetone carboxylation and suggest a role for manganese in nucleotide binding and activation.Acetone is a toxic molecule that is produced biologically by the fermentative metabolism of certain anaerobic bacteria and from ketone body breakdown in mammals (1, 2). In addition to producing acetone, mammals and microorganisms also metabolize acetone, producing acetoacetate or acetol (1-hydroxyacetone) from the carboxylation or hydroxylation, respectively, of acetone (3-12). Despite more than 60 years of research on the subject and direct evidence for enzymatic conversion of acetone to acetoacetate and acetol, the physiological significance of acetone metabolism in mammals remains unclear (2, 13). The role of acetone metabolism in bacteria is more clearly defined, in that a variety of aerobic and anaerobic bacteria are able to grow using acetone as their primary source of carbon and energy (14).Despite some early evidence suggesting that acetol is an intermediate in aerobic acetone metabolism by some bacteria (4,6,15), it now appears that carboxylation of acetone to acetoacetate is the primary, if not only, reaction by which both aerobic and anaerobic bacteria initiate acetone catabolism ...

Epoxyalkane:CoM transferase (EaCoMT) is a key enzyme of bacterial propylene metabolism, catalyzing the nucleophilic attack of coenzyme M (CoM, 2-mercaptoethanesulfonic acid) on epoxypropane to form the thioether conjugate 2-hydroxypropyl-CoM. The biochemical and molecular properties of EaCoMT suggest that the enzyme belongs to the family of alkyltransferase enzymes for which Zn plays a key role in activating an organic thiol substrate for nucleophilic attack on an alkyl-donating substrate. In the present work, the role of Zn in the EaCoMT-catalyzed reactions is established by removing Zn from EaCoMT, resulting in loss of catalytic activity that was restored upon addition of Zn back to the enzyme, and by expressing an inactive and Zn-deficient form of the enzyme that was activated by addition of ZnCl(2) or CoCl(2). Site-directed mutagenesis of one of the predicted Zn ligands (C220A) resulted in the formation of a largely catalytically inactive protein (0.06% of wild-type activity) that, when purified, contained a substoichiometric complement of Zn. EaCoMT was kinetically characterized and found to follow a random sequential mechanism with kinetic parameters K(m,epoxypropane) = 1.8 microM, K(m,CoM) = 34 microM, and k(cat) = 6.5 s(-1). The CoM analogues 2-mercaptopropionate, 2-mercaptoethanol, and cysteine substituted poorly for CoM as the thiol substrate, with specific rates of epoxyalkane conjugation that were at best 0.6% of the CoM-dependent rate, while ethanethiol, propanethiol, glutathione, homocysteine, and lipoic acid provided no activity. 2-Mercaptoethanol was a weak competitive inhibitor vs CoM with a K(I) of 192 mM. Isothermal titration calorimetry was used to investigate the thermodynamic binding determinants for the interaction of CoM and analogues with holo, Zn-deficient, and C220A EaCoMT variants. The stoichiometry of CoM binding correlated directly with the Zn content rather than monomer content of protein samples, reinforcing the importance of Zn in CoM binding. The binding of CoM to EaCoMT occurred with DeltaG = -7.5 kcal/mol (K(d) = 3.8 microM) and was driven by a large release of enthalpy. The thermodynamic contributors (K(a), DeltaG, DeltaH, DeltaS) to the individual binding of CoM, ethanesulfonate, and ethanethiol were determined and used to assess the contributions of the thiol, alkyl, and sulfonate moieties to total binding energy in the E x CoM binary complex.