Characterization of adenosine nucleosidase from Lupinus lutens L. Mcdonald, Nancy en_US
dc.contributor.department Chemistry en_US 2014-06-20T16:28:32Z 2014-06-20T16:28:32Z 1998 en_US
dc.description Adviser: Paul Kline. en_US
dc.description.abstract Adenosine nucleosidase (EC, a key enzyme in metabolism of purines and plant growth regulators, catalyzes the irreversible hydrolysis of adenosine to yield adenine and ribose. Adenosine nucleosidase had previously been isolated by Abusamhadneh and obtained in a high state of purity from yellow lupin seeds after four days of germination. A final purification step using an {dollar}\omega{dollar}-aminohexyl agarose column resulted in an enzyme of sufficient purity for further characterization of the enzyme. en_US
dc.description.abstract Adenosine nucleosidase was characterized utilizing several experimental approaches. One method was the measurement of kinetic isotope effects (KIEs) using stable isotopes of adenosine with the label in the ribose moiety. Kinetic isotope effects determined for (1{dollar}\sp\prime{dollar}-{dollar}\sp{lcub}13{rcub}{dollar}C) and (1{dollar}\sp\prime{dollar}-{dollar}\sp2{dollar}H) adenosine using gas chromatography/mass spectrometry (GC/MS) were 1.012 {dollar}\pm{dollar} 0.045 and 1.081 {dollar}\pm{dollar} 0.028 respectively. These KIEs are the average of three line pairs at 158/159, 187/188, and 217/218. The low values may indicate kinetic suppression. The BEBOVIB-IV program was used to calculate kinetic isotope effects to match the experimentally determined values. These calculations indicated an early transition state characterized by substantial bond order to the C1{dollar}\sp\prime{dollar}-N9 bond. en_US
dc.description.abstract Commitment to catalysis was determined using the pulse-chase method. The low ratio of product formed to substrate released indicate that the observed isotope effects are intrinsic. Substrate-trapping showed an initial burst followed by a slower rate of product formation. en_US
dc.description.abstract Certain structural features in the substrate are crucial to activity. Purines lacking an exocyclic nitrogen in the 6-position, a nitrogen in the 7-position, or a hydroxyl group in the C3{dollar}\sp\prime{dollar} position are poor substrates or do not react. The best substrate was 5{dollar}\sp\prime{dollar}-deoxyadenosine. en_US
dc.description.abstract The hydrolytic reaction can be reversed to give an experimental equilibrium constant of 263 M. Hydrolysis is preferred over synthesis. en_US D.A. en_US
dc.publisher Middle Tennessee State University en_US
dc.subject.lcsh Botanical chemistry en_US
dc.subject.lcsh Lupines en_US
dc.subject.lcsh Adenosine en_US
dc.subject.lcsh Nucleosidases en_US
dc.subject.lcsh Chemistry, Biochemistry en_US
dc.thesis.degreegrantor Middle Tennessee State University en_US
dc.thesis.degreelevel Doctoral en_US
dc.title Characterization of adenosine nucleosidase from Lupinus lutens L. en_US
dc.type Dissertation en_US
Original bundle
Now showing 1 - 1 of 1
No Thumbnail Available
2.86 MB
Adobe Portable Document Format