INTERMEDIATE FORMATION PROCESS IN THERMOLYSIN CATALYSIS OBSERVED USING A FLUORESCENT DISPLACEMENT PROBE IN THE STOPPED-FLOW METHOD

The thermolysin-catalyzed hydrolysis of N-Cbz-tripeptides was studied by means of a fluorescence stopped-flow method using N-dansyl-L-phenyl alanine (Dns-Phe) as a displacement probe at lower pH. To determine th e probe features of Dns-Phe, the equilibrium and kinetics of the binar y interaction between Dns-Phe and the enzyme were studied by means of static differential fluorescence and by fluorescence temperature-jump methods in the pH range 4.5-5.6. The dissociation constant (K-d) incre ased along with an increase in pH. Both the decreasing (apparent) bind ing rate and the increasing (apparent) dissociation rate with increasi ng pH were attributed to this decline of complex formation at higher p H. The pre-steady state process in the substrate hydrolytic reaction w as studied with this displacement probe, and the observed rate constan t for the four tripeptide substrates had saturation kinetics that indi cated the existence of a new intermediate during the sequence of the e nzyme reaction. Four tripeptide substrates had comparable k(2) values (rate constant of intermediate formation), whereas the K-S value of Cb z-Phe-Leu-Ala was about twice that of the others.