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(Hydroxyalkyl)cob(III)alamins as Competitive Inhibitors in Coenzyme B 12 ‐Dependent Enzymic Reactions: 1 H‐NMR Structure Analysis and Kinetic Studies with Glycerol Dehydratase and Diol Dehydratase
Author(s) -
Poppe László,
Hull William E.,
Nitsche Rainer,
Graf Torsten,
Stupperich Erhard,
Rétey János
Publication year - 1999
Publication title -
helvetica chimica acta
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.74
H-Index - 82
eISSN - 1522-2675
pISSN - 0018-019X
DOI - 10.1002/(sici)1522-2675(19990804)82:8<1250::aid-hlca1250>3.0.co;2-i
Subject(s) - chemistry , hydroxocobalamin , stereochemistry , dehydratase , enzyme , cofactor , non competitive inhibition , adenosylcobalamin , biochemistry , cyanocobalamin , vitamin b12
A series of (hydroxyalkyl)cobalamins, i.e. , 1a – d , (HO−(CH 2 ) n −Cbl, n =2 – 5), two diastereoisomeric (2,3‐dihydroxypropyl)cobalamins, i.e. , 2a , b ([( R )‐ and ( S )‐[(HO) 2 pr]‐Cbl) and their diastereoisomeric `base‐off' analogues, the ( Co β ‐2,3‐dihydroxypropyl‐[1′‐ O ‐( p ‐tolyl)cobamides]) 3a , b ([( R )‐ and ( S )‐(HO) 2 pr]‐PTC) were prepared and characterized by their 500‐MHz 1 H‐NMR spectra. The inhibitory activities of these compounds and of hydroxocobalamin (HO−Cbl) and (Co α ‐cyano)(Co β ‐hydroxo)[1′‐ O ‐( p ‐tolyl)cobamide] (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 and dihydroxypropyl derivatives of cobalamin acted as strong competitive inhibitors of coenzyme B 12 (5′‐deoxy‐5′‐adenosylcobalamin, Ado−Cbl) for 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−(CH 2 ) 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−(CH 2 ) n −Cbl ( 1c , d ; n =4, 5) were less efficient inhibitors, with [( S )‐(HO) 2 pr]−Cbl ( 2a ) performing 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‐alkylated analogue (HO,CN)−PTC proved to be a very poor inhibitor. These results confirm that the `base‐on' binding mode of coenzyme B 12 is preferred for GDH and DDH. The increase in K i 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.