(Hydroxyalkyl)cob(III)alamins as Competitive Inhibitors in Coenzyme B12-Dependent Enzymic Reactions:1H-NMR Structure Analysis and Kinetic Studies with Glycerol Dehydratase and Diol Dehydratase

Abstract

A series of (hydroxyalkyl)cobalamins, i.e., 1a – d, (HO−(CH2)n−Cbl, n=2 – 5), two diastereoisomeric (2,3-dihydroxypropyl)cobalamins, i.e., 2a,b ([(R)- and (S)-[(HO)2pr]-Cbl) and their diastereoisomeric `base-off' analogues, the (Coβ-2,3-dihydroxypropyl-[1′-O-(p-tolyl)cobamides]) 3a,b ([(R)- and (S)-(HO)2pr]-PTC) were prepared and characterized by their 500-MHz 1H-NMR spectra. The inhibitory activities of these compounds and of hydroxocobalamin (HO−Cbl) and (Coα-cyano)(Coβ-hydroxo)[1′-O-(p-tolyl)cobamide] (HO−PTC) were tested with two coenzyme-B12-dependent enzymes: glycerol dehydratase (GDH) and propane-1,2-diol dehydratase (DDH) (Table 4). The hydroxyalkyl and dihydroxypropyl derivatives of cobalamin acted as strong competitive inhibitors of coenzyme B12 (5′-deoxy-5′-adenosylcobalamin, Ado−Cbl) for both enzymes, with Ki values falling within the range defined by HO−Cbl (best inhibitor) and CN−Cbl (Ki /Km ratio of ca. 2). The short-chain HO−(CH2)n−Cbl (1a,b; n=2 or 3) exhibited Ki equal to the Km for Ado−Cbl. The [(R)- and (S)-(HO)2pr]−Cbl (2a,b) and the long-chain HO−(CH2)n−Cbl (1c,d; n=4, 5) were less efficient inhibitors, with [(S)-(HO)2pr]−Cbl (2a) performing slightly better than the (R)-diastereoisomer 2b for both enzymes. The `base-off' analogues, Ado−PTC and [(R)- and (S)-(HO)2pr]−PTC (3a,b), were moderate inhibitors with Ki/Km ratios of 4.5 – 28 for GDH or 7 – 13 for DDH. [(S)-(HO)2pr]−PTC (3a) was the best inhibitor in this group. The non-alkylated analogue (HO,CN)−PTC proved to be a very poor inhibitor. These results confirm that the `base-on' binding mode of coenzyme B12 is preferred for GDH and DDH. The increase in Ki for PTC- vs. Cbl-type inhibitors may result from the entropic penalty required for folding of the PTC nucleotide chain into a Cbl-like loop conformation. Hydrophilic interactions between the β-ligand of the inhibitor and ribosyl- or substrate-binding sites may make an important contribution to the formation or stabilization of the apoenzyme-inhibitor complex, especially for the PTC derivative.