The steady state and pre-steady state kinetics of hydrolysis of the fluorescent peptide substrate, Leu-Gly-NHNH-Dns, by porcine kidney leucine aminopeptidase (LAP) have been studied by stopped flow cryoenzymology. Experiments have been carried out with LAP species containing Zn(II) in the catalytic site and various other divalent metal ions in the regulatory site. These species are denoted ((LAP)Zn$\sb6$Me$\sb6$), where Me(II) = Zn(II), Ni(II), Cu(II), Mg(II), Mn(II) and empty. The stopped flow fluorescence traces obtained with direct substrate excitation for these reactions carried out at ambient temperatures are consistent with formation of a single intermediate, (E$\cdot$S), and have been used to determine the steady-state kinetic parameters. When the metal ion in the regulatory site is changed, k$\sb{\rm cat}$ and K$\sb{\rm M}$ are altered in the same direction to approximately the same extent, leaving the values of k$\sb{\rm cat}$/K$\sb{\rm M}$ almost unchanged. / The effect of methanol on the hydrolysis of Leu-Gly-NHNH-Dns by ((LAP)Zn$\sb6$Me$\sb6$) has also been investigated. Increasing percentages of methanol lower k$\sb{\rm cat}$ markedly and decrease K$\sb{\rm M}$ slightly, causing a net decrease in k$\sb{\rm cat}$/K$\sb{\rm M}$. Low-temperature stopped-flow experiments for the reaction of Leu-Gly-NHNH-Dns with ((LAP)Zn$\sb6$Me$\sb6$) have been carried out in 50% methanol, 0.1 M KCl, 10 mM Tris, pH$\sp*$ 9.0 under non-turnover conditions. For several ((LAP)Zn$\sb6$Me$\sb6$) a new relaxation is observed at $-40\sp\circ$C that has been shown to correspond to the interconversion of a new intermediate, (E$\cdot$S) $\sp\prime$, with (E$\cdot$S). The pre-steady state kinetic parameters, K$\sb{\rm s}$, k$\sb2$ and k$\sb{-2}$, for the hydrolysis of Leu-Gly-NHNH-Dns by ((LAP)Zn$\sb6$Ni$\sb6$) at $-40\sp\circ$C are 0.28 mM, 54 sec$\sp{-1}$ and 4.4 sec$\sp{-1}$, respectively. / Source: Dissertation Abstracts International, Volume: 48-09, Section: B, page: 2658. / Thesis (Ph.D.)--The Florida State University, 1987.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_76161 |
| Contributors | LIN, WANN-YIN., Florida State University |
| Source Sets | Florida State University |
| Detected Language | English |
| Type | Text |
| Format | 287 p. |
| Rights | On campus use only. |
| Relation | Dissertation Abstracts International |
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