Characterization of adenosine nucleosidase from Lupinus lutens L.

Abstract

Adenosine nucleosidase (EC 3.2.2.7), 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.

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.

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.

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.

The hydrolytic reaction can be reversed to give an experimental equilibrium constant of 263 M. Hydrolysis is preferred over synthesis.