Structure-activity relationship in the urokinase hydrolysis of alpha-N-acetyl-L-lysine anilides.
The absence of both nonproductive binding and substrate activation and also the good solubility of the substrates make the urokinase-catalysed hydrolysis of specific anilides a very suitable reaction for substrate structure-enzyme activity studies. Derivatives of alpha-N-acetyl-L-lysine anilide with high sigma minus-value substituents in the aniline ring were synthesized. Rate constants kappa-cat. and apparent Michaelis-Menten constants K-m (app.) are presented. From the substituent dependence of kappa-cat. and from the fact that kappa-cat. is 13 to 37 times smaller than the deacylation rate constant it is concluded that the rate-limiting step proceeds prior to deacylation. The catalytic rate constant kappa-cat. obeys a linear free-energy relationship of the Hammett type with Q equals +0.72. Two different mechanisms implied by the results obtained from the model reaction (specific base and general acid-base catalysed hydrolysis of N-acetylglycine anilides under extreme conditions) are proposed in order to account for this positive and low Q-value. In the first mechanism the breakdown of an enzyme tetrahedral intermediate is rate-limiting, while in the second one its formation controls the overall rate. The discrimination between the two mechanisms, however, could not be found.
D000813:Anilides / Q000378:metabolism*; D001665:Binding Sites; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D013329:Structure-Activity Relationship; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D000813:Anilides; D010450:Endopeptidases; D014568:Urokinase-Type Plasminogen Activator; D008239:Lysine