Reactions between dipeptidyl peptidase IV and diacyl hydroxylamines: mechanistic investigations

• Published in: Journal of enzyme inhibition Vol. 2; no. 4; p. 239
• Main Authors: Demuth, H U, Neumann, U, Barth, A
• Format: Journal Article
• Language: English
• Published: Switzerland 1989
• Subjects: Dipeptidyl Peptidase 4; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases - antagonists & inhibitors; Hydroxylamines - pharmacology; Kinetics; Protein Binding; Spectrophotometry, Ultraviolet - methods; Structure-Activity Relationship; Thermodynamics
• ISSN: 8755-5093
• DOI: 10.3109/14756368909088477

Kinetics of inactivation of dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) by N-peptidyl-O-(4-nitrobenzoyl) hydroxylamines and their enzyme-catalyzed hydrolysis were followed using independent monitoring methods, all giving similar efficiency ratios of Kcat/Kinact. Different temperature dependences of the DP IV-inactivation and enzyme-catalyzed hydrolysis provide evidence of independent rate determining steps for both reactions. Activation parameters of inactivation are similar to those of spontaneous decomposition of the compounds, suggesting a mechanistic relationship. Investigation of DP IV-inactivation, DP IV-catalyzed hydrolysis of N-Ala-Pro-O-Bz(4-NO2) and the decomposition of the suicide substrate in H2O and D2O gave solvent isotope effects of 4.65, 2.54 and 1.02, respectively. A proton inventory of the inactivation reaction indicates involvement of more than one proton in the formation or breakdown of its transition state. The linear proton inventory found for the hydrolytic reaction is consistent with one proton transition in the rate determining step and resembles the rate limiting deacylation of Ala-Pro-DP IV. The hypothetical reaction model now locates splitting in both reactions prior to formation of a covalent intermediate during the catalytic cycle.