(Hydroxyalkyl)cob(III)alamins as competitive inhibitors in coenzyme B-12-dependent enzymic reactions: H-1-NMR structure analysis and kinetic studies with glycerol dehydratase and diol dehydratase

A series of (hydroxyalkyl)cobalamins, Lc. 1a-d, (HO-(CH2)(n)-Cbl, n=2-5), t wo diastereoisomeric (2.3-dihydroxypropyl)cobalamins ie.,2a;b ([(R)- and (S )-[(HO)(2)pr]-Cbl) and their diastereoisomeric 'baseoff' analogues, the (Co beta-2,3-dihydroxypropyl-[t'-O-( p-tolyl)cobamides]) 3a,b ( It R)- and ( S )-( IIO)(2)pr]-PTC) were prepared and characterized by their 500-MHz H-1-NM R spectra. The inhibitory activities of these compounds and of hydroxocobal amin (HO-Cbl) and (Co alpha-cyano)(Co beta-hydroxo)[1'-O-(p-tolyl) (HO-PTC) were tested with two coenzyme-B-12-dependent enzymes: glycerol dehydratase (GDH) and propane-1,2-diol dehydratase (DDH) (Table 4). The hydroxyalkyl a nd dihydroxypropyl derivatives of cobalamin acted as strong competitive inh ibitors of coenzyme B-12 ( 5'-deoxy-5'-adenosylcobalamin, Ado-Cbl) fur both enzymes. with K-i values falling within the range defined by HO -Cbl (best inhibitor) and CN-Cbl (K-i/K-m ratio of ca. 2). The short-chain HO-(CH2)(n )-Cbl (1a,b; n=2 or 3) exhibited K-i equal to the K-m for Ado-Cbl. The [(R) - and (S)-(HO)(2)pr] -Cbl (2a,b) and the long-chain HO-(CH2)(n)-Cbl (1c,d;n =4,5) were less efficient inhibitors, with [(S)-(HO)(2)pr]- Cbl (2a) perfor ming slightly better than the (R)-diastereoisomer 2b for both enzymes. The 'base-off analogues, Ado-PTC and [(R)- and (S)-(HO)(2)pr]-PTC (3a,b), were moderate inhibitors with K-i/K-m ratios of 4.5-28 for GDH or 7-13 for DDH. [(S)-(HO)(2)pr]-PTC (3a) was the best inhibitor in this group. The non-alky lated analogue ( HO,CN)- PTC proved to be a very poor inhibitor. These resu lts confirm that the 'base-on' binding mode of coenzyme B-12 is preferred f or GDH and DDH. The increase in K-i for PTC- vs. Cbl-type inhibitors may re sult from the entropic penalty required for folding of the PTC nucleotide c hain into a Cbl-like loop conformation. Hydrophilic interactions between th e beta-ligand of the inhibitor and ribosyl- or substrate-binding sites may make an important contribution to the formation or stabilization of the apo enzyme-inhibitor complex, especially for the PTC derivative.