The flavoprotein spermine oxidase catalyzes the oxidation of spermine and oxygen to spermidine, 3-aminopropanol, and hydrogen peroxide. To allow mechanistic studies of the enzyme, methods have been developed to obtain large amounts of purified recombinant protein. The enzyme requires co-expression with chaperone proteins GroEL and GroES to remain soluble and active. Purification requires the use of a Ni-NTA and size exclusion column. Human spermine oxidase is a monomer with an extinction coefficient of 14000 M-1cm-1. The kinetic mechanism is ping pong. Therefore, oxygen is bound to the enzyme before spermidine is released. N1-Acetyl spermine is a slow substrate with kcat and kcat/Km values 2 and 3 orders of magnitude smaller than the values for spermine. Spermidine is a competitive inhibitor, and 1,8-diaminooctane (DAO) is an uncompetitive inhibitor. The pH effects indicate that two ionizable groups are present in the kcat/Km profile and one ionizable group is in the kcat profile. The reductive half reaction reveals no phase other than the reduction of the FAD, indicating the probability of a single chemical step. Reduction is not limiting to the overall reaction. Isotope effects were determined; Dkcat at pH 7.5 = 4.1±0.4, pH 8.5 = 2.6±0.01.
| Identifer | oai:union.ndltd.org:TEXASAandM/oai:repository.tamu.edu:1969.1/ETD-TAMU-3108 |
| Date | 15 May 2009 |
| Creators | Juarez, Paul Ramon |
| Contributors | Fitzpatrick, Paul F., Scholtz, J. Martin, Shippen, Dorothy E. |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | thesis, text |
| Format | electronic, application/pdf, born digital |
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