Taurine/alpha-ketoglutarate dioxygenase (TauD), a member of the broad class of non-heme Fe(II) oxygenases, converts taurine (2-aminoethanesulfonate) to sulfite and aminoacetaldehyde while decomposing alpha-ketoglutarate (alphaKG) to form succinate and CO(2). Under anaerobic conditions, the addition of alphaKG to Fe(II)TauD results in the formation of a broad absorption centered at 530 nm. On the basis of studies of other members of the alphaKG-dependent dioxygenase superfamily, we attribute this spectrum to metal chelation by the substrate C-1 carboxylate and C-2 carbonyl groups. Subsequent addition of taurine perturbs the spectrum to yield a 28% greater intensity, an absorption maximum at 520 nm, and distinct shoulders at 480 and 570 nm. This spectral change is specific to taurine and does not occur when 2-aminoethylphosphonate or N-phenyltaurine is added. Titration studies demonstrate that each TauD subunit binds a single molecule of Fe(II), alphaKG, and taurine. In addition, these studies indicate that the affinity of TauD for alphaKG is enhanced by the presence of taurine. alpha-Ketoadipate, the other alpha-keto acid previously shown to support TauD activity, and alpha-ketocaproate lead to the formation of weak 520 nm-like spectra with Fe(II)TauD in the presence of taurine; however, corresponding spectra at 530 nm are not observed in the absence of taurine. Pyruvate and alpha-ketoisovalerate fail to elicit absorption bands in this region of the spectrum, even in the presence of taurine. Stopped-flow UV-visible spectroscopy reveals that the 530 and 520 nm spectra associated with alphaKG-Fe(II)TauD and taurine-alphaKG-Fe(II)TauD are formed at catalytically competent rates ( approximately 40 s(-)(1)). The rate of chromophore formation was independent of substrate or enzyme concentration, suggesting that alphaKG binds to Fe(II)TauD prior to the formation of a chromophoric species. Significantly, the taurine-alphaKG-Fe(II)TauD state, but not the alphaKG-Fe(II)TauD species, reacts rapidly with oxygen (42 +/- 9 s(-)(1)). Using the data described herein, we develop a preliminary kinetic model for TauD catalysis.

Methylmalonyl-CoA mutase catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA. It is dependent on the cofactor, coenzyme B12 or adenosylcobalamin, for activity. The first step in this, and other coenzyme B12-dependent reactions, is postulated to be homolysis of the Co-C bond of the cofactor. Methylmalonyl-CoA mutase accelerates the rate of Co-C bond homolysis by a factor of approximately 10(12). The strategy employed by the enzyme for the remarkable labilization of this bond is not known. Using UV-visible stopped-flow spectrophotometry, we demonstrate that the Co-C homolysis rate in the presence of protiated substrate has a rate constant of >600 s(-1) at 25 degrees C. In the presence of [CD3]methylmalonyl-CoA, this rate decreases to 28 +/- 2 s(-1). These results suggest that Co-C bond homolysis is coupled to hydrogen atom abstraction from the substrate and that the intrinsic binding energy of substrate may be a significant contributor to catalysis by methylmalonyl-CoA mutase.

The determination of its crystal structure, as well as extensive biochemical characterizaion, has focused attention on methylmalonyl-coenzymeA (MM-CoA) mutase as a paradigm for AdoCbl (adenosylcobalamin)-dependent enzymes. These enzymes catalyze carbon skeleton rearrangement reactions via a radical mechanism, which originates in homolysis of the Co−C bond. The determinants of this mechanism are of great chemical and biochemical interest. We report resonance Raman (RR) spectra of MM-CoA, in the presence of substrates and inhibitors, using a cryogenic technique to prevent laser-induced photolysis of the Co−C bond. RR spectroscopy provides an in-site probe of cobalamin structure. Although the spectra are dominated by the corrin ring, four RR bands arising from Co-bound adenosine can be detected via isotope editing. These are assigned to the Co−C bond stretch, the Co−C−C angle bend, a 5‘-C-coupled ribose deformation, and a hindered rotation of the adenosine about the Co−C bond. The RR spectra confirm enzyme-catalyzed H exchange between substrate, but not inhibitors, and the adenosyl 5‘-C atom. The RR enhancement pattern is affected in different ways by binding inhibitors or slow substrates on the one hand and product or substrate on the other. Since product dissociation is apparently rate-limiting and the rearrangement equilibrium lies toward product, the adducts with product or substrate represent the product state (P state), whereas those with inhibitors or the slow substrates represent the substrate state (S state). The RR enhancement changes (activation of δCoCC and weakening and downshift of νCoC) indicate Co-Ado tilting, to a small extent in the resting enzyme and to a larger extent in the S state. These changes are reversed in the P state. Thus Co-Ado tilting is identified as a contributor to activation of the Co−C bond in the S state. The steric forces that induce tilting are apparently relaxed in the P state, thus promoting the rearrangement of substrate to product. The corrin vibrational modes are responsive to the ring conformation but are mostly unaffected by substrate or inhibitor binding, indicating that Co−C activation does not involve corrin conformation changes. However, two modes, whose frequencies (423 and 437 cm-1) are consistent with contributions from Co−N(corrin) stretching, shift down in the P state, suggesting displacement of the Co from the corrin plane. This effect may result from lengthening of the Co−N(histidine) bond, as seen in the crystal structure.

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