Characterization of recombinant human renin: Kinetics,pH-stability, and peptidomimetic inhibitor binding |
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| Authors: | Thomas F. Holzman, Christine C. Chung, Rohinton Edalji, David A. Egan, Margaret Martin, Earl J. Gubbins, Grant A. Krafft, Gary T. Wang, A. Mitchel Thomas Saul H. Rosenberg etAlia" >,et al. |
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| Affiliation: | (1) Protein Biochemistry, Pharmaceutical Discovery Research, Pharmaceutical Products Division, Abbott Laboratories, 60064 Abbott Park, Illinois;(2) Molecular Biology, Pharmaceutical Discovery Research, Pharmaceutical Products Division, Abbott Laboratories, 60064 Abbott Park, Illinois;(3) Probe Molecular Design, Abbott Diagnostics Division, Abbott Laboratories, 60064 Abbott Park, Illinois;(4) Drug Design and Delivery, Pharmaceutical Discovery Research, Pharmaceutical Products Division, Abbott Laboratories, 60064 Abbott Park, Illinois;(5) Cardiovascular Research, Pharmaceutical Discovery Research, Pharmaceutical Products Division, Abbott Laboratories, 60064 Abbott Park, Illinois;(6) Computer-Aided Molecular Design, Pharmaceutical Discovery Research, Pharmaceutical Products Division, Abbott Laboratories, 60064 Abbott Park, Illinois;(7) Present address: Department of Biochemistry and Biophysics, Yale University, New Haven, Connecticut |
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| Abstract: | The kinetic behavior andpH-stability of recombinant human renin was analyzed using a new fluorogenic substrate based on the normal P6-P3 renin cleavage sequence in human angiotensinogen. The design of this fluorogenic substrate makes possible, for the first time, direct monitoring of the kinetics of proteolytic conversion of prorenin to renin. ThepH-stability profile for renin, measured with the substrate at 25°C, indicated a broad plateau of stability betweenpH 6.0 and 10.0. Analysis of thepH-activity profile of renin for the substrate indicated a minimumKm ( 1.8 µM) atpH 7.4 and a maximumVm betweenpH 7.4 and 8.0. The thermodynamics of the binding of a novel, soluble, peptidomimetic inhibitor to renin indicated it is possible to retain the tight-binding characteristics and enthalpy contributions to binding of larger peptide-derived inhibitors, while reducing inhibitor size and entropic contributions to binding. A novel derivative of the fluorogenic substrate, containing a 3-methyl histidine substitution at the P2 site, was used to test the recent hypothesis that renin functions by virtue of substrate-directed catalysis. |
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| Keywords: | Renin inhibitors fluorescence calorimetry substrate-directed catalysis |
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