Abstract
P. Galuszka et al proposed that cytokinin oxidase(CKO) behaves as a dehydrogenase rather than an oxidase. We examine the mode of the side-chain cleavage by CKO using deuterium-labeled iPA.
CKO was extracted from Agrobacterium-transformed callus of sweet potato, and partially purified. CKs used as substrates were α-methyl-iPAs and deuterated iPAs that deuterium was substituted for hydrogen at α-methylene(d2) and at γ,γ-dimethyl(d6) of the side chain. The reaction rate was determined on the base of HPLC analysis.
When α-methyl-iPAs were used as substrates, no both the enantiomers were degraded by CKO. It seems that methylation at α position of the side-chain rendered the CK resistant to CKO.
Deuterium substitution at α-methylene revealed a KiPA/KiPA-d2=3.5 and on the dimethyl group showed a KiPA/KiPA-d6=0.7. The results are explained in terms of both primary and secondary isotope effects on a possible rate-determining step in the side-chain cleavage.
