A Kinetic Study of the Hydrolysis of theN-Tosylalanine Ester of 3-Hydroxy-5-phenylpyrrole and Related Compounds by Human Leukocyte Elastase

The tosylalanine ester of 3-hydroxy-5-phenylpyrrole (HOPPy) is localized on reagent strips (Ames LEUKOSTIX) and used diagnostically to test urine for the presence of human leukocyte elastase (HLE) as an indication of urinary tract infection. We have determined the kinetic constants for the HLE-catalyzed hydrolysis of this substrate and the related substrates Tos-Ala-ONp, Cbz-Ala-OPPy, and Cbz-Ala-ONp, in solution at three different pH values. In the reagent strip matrix, diazo coupling of 4-diazo-3-hydroxy-1-napthylsulfonate[formula]with the enzymatic hydrolysis product HOPPy generates a purple color. We have also studied the kinetics of the reaction of HOPPy with[formula]and other related diazonium salts such as 4-diazo-3-hydroxy-7-nitro-1-napthylsulfonate, 4-diazo-3-hydroxy-1,7-napthyldisulfonate, and 2-methoxy-4-(N-morpholinyl)benzene diazonium chloride. Tos-Ala-OPPy is the most reactive substrate among the compounds examined and kinetic studies indicate that deacylation is rate-limiting for HLE hydrolysis. The presence of decanol accelerates the enzymatic hydrolysis of Tos-Ala-OPPy with akcat/KM= 107M−1s−1, which is close to the diffusion-controlled limit. For the diazo coupling reaction, the rate is affected by the substituents on the naphthalene ring and by the buffer in which the reaction occurs. This research has elucidated some important mechanistic features for the reaction of these compounds and may lead to improved methods for the detection of leukocyte elastase.