Thermolysin is a thermostable neutral zinc endopeptidase with a molecular mass of 34.6 kDa. The stopped-flow fluorescence technique has been used to investigate the catalytic pathway for the hydrolysis of dansyl-peptides by thermolysin. The origin of the fluorescence changes observed during the hydrolysis of dansyl-substrates by thermolysin has been investigated. Depending on the substrate, these changes arise from changes in the concentration of the substrate (F$\sp{\rm SP}$) or the changes in the concentration of ES$\sb{\rm i}$ as reflected by resonance energy transfer (F$\sp{\rm RET}$). Both F$\sp{\rm SP}$ and F$\sp{\rm RET}$ changes have been used to monitor the reaction process, but different mathematical formalisms have been used to determine the kinetic parameters and the results from all are in good agreement. The F$\sp{\rm RET}$ contribution correlates strongly with the K$\sb{\rm M}\sp{-1}$ values for these reactions, as expected for a process that monitors the (ES$\sb{\rm i}$). The effect of sodium halide salts on the optical and emission properties of thermolysin and its dansyl substrates has been studied. The effect of these salts on the activity of thermolysin is substrate dependent. The main effect is to reduce the K$\sb{\rm M},$ indicating that chloride increases the strength of substrate binding. For some substrates, k$\sb{\rm cat}$ is compensatorily reduced, while for others it is unchanged. The effects of these salts can be described by a general modifier mechanism. Low-temperature stopped-flow fluorescence measurements have been carried out to study elementary steps that occur during the pre-steady-state time region reaction of thermolysin with Dns-Gly-Phe-Ala. A new relaxation characterized by an increase in dansyl fluorescence and decrease in Trp fluorescence has been observed. This corresponds either to an isomerization of thermolysin or to the interconversion of two ES$\sb{\rm i}.$ / Source: Dissertation Abstracts International, Volume: 53-11, Section: B, page: 5706. / Major Professor: Harold E. Van Wart. / Thesis (Ph.D.)--The Florida State University, 1992.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_76823 |
| Contributors | Yang, Jenny Jie., Florida State University |
| Source Sets | Florida State University |
| Language | English |
| Detected Language | English |
| Type | Text |
| Format | 221 p. |
| Rights | On campus use only. |
| Relation | Dissertation Abstracts International |
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