文摘
Maize polyamine oxidase (MPAO) is a flavin adenine dinucleotide (FAD)-dependent enzymethat catalyses the oxidation of spermine and spermidine at the secondary amino groups. The structure ofMPAO indicates a 30-Å long U-shaped tunnel that forms the catalytic site, with residues Glu62 andGlu170 located close to the enzyme-bound FAD and residue Tyr298 in close proximity to Lys300, whichin turn is hydrogen-bonded to the flavin N5 atom via a water molecule (HOH309). To provide insightinto the role of these residues in the catalytic mechanism of FAD reduction, we have performed steady-state and stopped-flow studies with wild-type, Glu62Gln, Glu170Gln, Tyr298Phe, and Lys300Met MPAOenzymes. We show that the steady-state enzyme activity is governed by an ionisable group with amacroscopic pKa of ~5.8. Kinetic analysis of the Glu62Gln, Glu170Gln, and Tyr298Phe MPAO enzymeshave indicated (i) only small perturbations in catalytic activity as a result of mutation and (ii) steady-statepH profiles essentially unaltered when compared to the wild-type enzyme, suggesting that these residuesdo not play a critical role in the reaction mechanism. These kinetic observations are consistent withcomputational calculations that suggest that Glu62 and Glu170 are protonated over the pH range accessibleto kinetic studies. Substitution of Lys300 with Met in MPAO resulted in a 1400-fold decrease in the rateof flavin reduction and a 160-fold decrease in the equilibrium dissociation constant for the Lys300Met-spermidine complex, consistent with a major role for this residue in the mechanism of substrate oxidation.A sizable solvent isotope effect (SIE = 5) accompanies FAD reduction in the wild-type enzyme andsteady-state turnover (SIE = 2.3) of MPAO, consistent with the reductive half-reaction of MPAO makinga major contribution to rate limitation in steady-state turnover. Studies using the enzyme-monitored turnovermethod indicate that oxidized FAD is the prominent form during steady-state turnover, consistent withthe reductive half-reaction being rate-limiting. Our studies indicate the importance of Lys300 and probableimportance of HOH309 to the mechanism of flavin reduction in MPAO. Possible roles for Lys300 andwater in the mechanism of flavin reduction are discussed.
