Observation of the pre-steady state process in thermolysin catalysis with a fluorescent displacement probe at low pH.
The pre-steady state process in the thermolysin-catalyzed hydrolysis of Cbz-Gly-Phe-Ala was observed at pH 4.5 by fluorescence stopped-flow method using Dns-Phe as a displacement probe. After the confirmation of the pre-equilibrium hypothesis for the binary interaction, the nonlinear substrate concentration dependence of the apparent kinetic constant for the pre-steady state process was analyzed and an existence of multi-intermediates was proposed.
Kunugi
S
S
Department of Polymer Science and Engineering, Kyoto Institute of Technology, Japan.
Yokoyama
M
M
Sakamoto
A
A
D000595:Amino Acid Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013820:Thermolysin / Q000378:metabolism*
D005456:Fluorescent Dyes; D009842:Oligopeptides; D013820:Thermolysin