Molecular clip and tweezer introduce new mechanisms of enzyme inhibition.
Artificial molecular clips and tweezers, designed for cofactor and amino acid recognition, are able to inhibit the enzymatic activity of alcohol dehydrogenase (ADH). IC50 values and kinetic investigations point to two different new mechanisms of interference with the NAD(+)-dependent oxidoreductase: While the clip seems to pull the cofactor out of its cleft, the tweezer docks onto lysine residues around the active site. Both modes of action can be reverted to some extent, by appropriate additives. However, while cofactor depletion by clip 1 was in part restored by subsequent NAD(+) addition, the tweezer (2) inhibition requires the competitive action of lysine derivatives. Lineweaver-Burk plots indicate a competitive mechanism for the clip, with respect to both substrate and cofactor, while the tweezer clearly follows a noncompetitive mechanism. Conformational analysis by CD spectroscopy demonstrates significant ADH denaturation in both cases. However, only the latter case (tweezer-lysine) is reversible, in full agreement with the above-detailed enzyme switch experiments. The complexes of ADH with clips or tweezer can be visualized in a nondenaturing gel electrophoresis, where the complexes migrate toward the anode, in contrast to the pure enzyme which approaches the cathode. Supramolecular chemistry has thus been employed as a means to control protein function with the specificity of artificial hosts opening new avenues for this endeavor.
Talbiersky
Peter
P
Department of Chemistry, University Duisburg-Essen, 45117 Essen, Germany.
Bastkowski
Frank
F
Klärner
Frank-Gerrit
FG
Schrader
Thomas
T
D000426:Alcohol Dehydrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008968:Molecular Conformation; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D010755:Organophosphates / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D011084:Polycyclic Aromatic Hydrocarbons / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology
D004791:Enzyme Inhibitors; D010755:Organophosphates; D011084:Polycyclic Aromatic Hydrocarbons; D009243:NAD; D000426:Alcohol Dehydrogenase; D008239:Lysine