Dioldehydrase:  An Essential Role for Potassium Ion in the Homolytic Cleavage of the Cobalt−Carbon Bond in Adenosylcobalamin

Abstract

The complex of dioldehydrase with adenosylcobalamin (Coenzyme B 12 ) and potassium ion reacts with molecular oxygen in the absence of a substrate to oxidize coenzyme and inactivate the complex. In this paper, high performance liquid chromatography and mass spectral analysis are used to identify the nucleoside products resulting from oxygen inactivation. The product profile indicates that oxygen inactivation proceeds by direct reaction of molecular oxygen with the 5′-deoxyadenosyl radical and cob(II)alamin. Formation of 5′-peroxyadenosine as the initial nucleoside product chemically correlates this reaction with aerobic, aqueous photoinduced homolytic cleavage of adenosylcobalamin (Schwartz, P. A. and Frey, P. A., (2007) Biochemistry, accompanying paper), indicating that both reactions proceed through similar chemical intermediates. The oxygen inactivation of the enzyme-coenzyme complex shows an absolute requirement for the same monocations required in catalysis by dioldehydrase. Measurements of the dissociation constants for adenosylcobalamin from potassium-free (K d = 16 ± 2 μM) or potassium-bound dioldehydrase (K d = 0.8 ± 0.2 μM), reveal that the effect of the monocation in stimulating oxygen sensitivity cannot be explained by an effect on the binding of coenzyme to the enzyme. Cross-linking experiments suggest that the full quaternary structure is assembled in the absence of potassium ion under the experimental conditions. The results indicate that dioldehydrase likely harvests the binding energy of the activating monocation to stimulate the homolytic cleavage of the Co-C5′ bond in adenosylcobalamin.Dioldehydrase (DL-1,2-propanediol hydro-lyase, EC 4.2.1.28) catalyzes the adenosylcobalamin-dependent dehydration of 1,2-propanediol to propionaldehyde (1). Similar to other adenosylcobalamin-dependent enzymes, the reaction involves a hydrogen abstraction from substrate by the 5′-deoxyadenosyl radical generated through homolytic cleavage of the Co-C5′ bond in the coenzyme (Figure 1) (2). After substrate-radical generation by hydrogen abstraction, isomerization proceeds through the 1,2-migration of the hydroxyl moiety to the terminal carbon, where a hydroxyl group is subsequently eliminated to form the aldehyde. A minimal mechanism for the reaction of dioldehydrase with 1,2-propanediol is illustrated in Scheme 1.

Crystal Structures of Ethanolamine Ammonia-lyase Complexed with Coenzyme B12 Analogs and Substrates

“…Apparent substrate triggering of the Co-C bond cleavage would be due to the shift of equilibrium toward the formation of much more stable substrate radical. The Co-C bond cleavage of AdoCbl and 3Ј,4Ј-anhydro-AdoCbl by this enzyme in the absence of substrate, but in the presence of potassium ion, has been reported (52)(53)(54), although the cleavage rates are much slower than in the presence of substrate.…”

Metabolic functions of the human gut microbiota: the role of metalloenzymes

“…An essential K + ion binds in the protein active site near the adenine ring, inducing a protein conformational change and resulting in Co-C bond cleavage even in the absence of substrate. 111,112,115 The substrate coordinates a Ca 2+ ion via its two hydroxyl groups, thereby increasing its effective size and resulting in a larger energetic release upon binding, 111,112,116 which could balance the energetic cost of Co-C bond cleavage. 117 When the Co-C bond cleavage event occurs in the presence of substrate, the resultant 5 0 -dA c abstracts a hydrogen atom from the C1 position of (S)-1,2-propanediol to generate an a-hydroxyalkyl substrate radical (Fig.…”

Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme

“…However, in yet another parallel between the radical SAM and B 12 radical enzymes, assays of PFL-AE activity reveal that its catalytic function is controlled by the presence and identity of the bound monovalent cation, requiring a K + ion bound in the active site for optimal activity, as is the case for the B 12 enzyme dioldehydrase. 16 , 17 …”