The kinetic behavior and pH-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. The pH-stability profile for renin, measured with the substrate at 25 degrees C, indicated a broad plateau of stability between pH 6.0 and 10.0. Analysis of the pH-activity profile of renin for the substrate indicated a minimum Km (approximately 1.8 microM) at pH approximately 7.4 and a maximum Vm between pH 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.