Mechanistic and structural analyses of the roles of active site residues in yeast polyamine oxidase Fms1: characterization of the N195A and D94N enzymes.

Flavoprotein Fms1 from Saccharomyces cerevisiae catalyzes the oxidation of spermine in the biosynthetic pathway for pantothenic acid. The same reaction is catalyzed by the mammalian polyamine and spermine oxidases. The active site of Fms1 contains three amino acid residues positioned to interact with the polyamine substrate, His67, Asn195, and Asp94. These three residues form a hydrogen-bonding triad with Asn195 being the central residue. Previous studies of the effects of mutating His67 are consistent with that residue being important both for interacting with the substrate and for maintaining the hydrogen bonds in the triad [Adachi, M. S., Taylor, A. B., Hart, P. J., and Fitzpatrick, P. F. (2012) Biochemistry 51, 4888-4897]. The N195A and D94N enzymes have now been characterized to evaluate their roles in catalysis. Both mutations primarily affect the reductive half-reaction. With N(1)-acetylspermine as the substrate, the rate constant for flavin reduction decreases ~450-fold for both mutations; the effects with spermine as the substrate are smaller, 20-40-fold. The k(cat)/K(amine)- and k(cat)-pH profiles with N(1)-acetylspermine are only slightly changed from the profiles for the wild-type enzyme, consistent with the pK(a) values arising from the amine substrate or product and not from active site residues. The structure of the N195A enzyme was determined at a resolution of 2.0 Å. The structure shows a molecule of tetraethylene glycol in the active site and establishes that the mutation has no effect on the protein structure. Overall, the results are consistent with the role of Asn195 and Asp94 being to properly position the polyamine substrate for oxidation.

Mechanistic and structural analyses of the role of His67 in the yeast polyamine oxidase Fms1.

The flavoprotein oxidase Fms1 from Saccharomyces cerevisiae catalyzes the oxidation of spermine and N(1)-acetylspermine to spermidine and 3-aminopropanal or N-acetyl-3-aminopropanal. Within the active site of Fms1, His67 is positioned to form hydrogen bonds with the polyamine substrate. This residue is also conserved in other polyamine oxidases. The catalytic properties of H67Q, H67N, and H67A Fms1 have been characterized to evaluate the role of this residue in catalysis. With both spermine and N(1)-acetylspermine as the amine substrate, the value of the first-order rate constant for flavin reduction decreases 2-3 orders of magnitude, with the H67Q mutation having the smallest effect and H67N the largest. The k(cat)/K(O2) value changes very little upon mutation with N(1)-acetylspermine as the amine substrate and decreases only an order of magnitude with spermine. The k(cat)/K(M)-pH profiles with N(1)-acetylspermine are bell-shaped for all the mutants; the similarity to the profile of the wild-type enzyme rules out His67 as being responsible for either of the pK(a) values. The pH profiles for the rate constant for flavin reduction for all the mutant enzymes similarly show the same pK(a) as wild-type Fms1, about ∼7.4; this pK(a) is assigned to the substrate N4. The k(cat)/K(O2)-pH profiles for wild-type Fms1 and the H67A enzyme both show a pK(a) of about ∼6.9; this suggests His67 is not responsible for this pH behavior. With the H67Q, H67N, and H67A enzymes the k(cat) value decreases when a single residue is protonated, as is the case with the wild-type enzyme. The structure of H67Q Fms1 has been determined at a resolution of 2.4 Å. The structure shows that the mutation disrupts a hydrogen bond network in the active site, suggesting that His67 is important both for direct interactions with the substrate and to maintain the overall active site structure.

Mechanistic studies of the yeast polyamine oxidase Fms1: kinetic mechanism, substrate specificity, and pH dependence.

The flavoprotein oxidase Fms1 from Saccharomyces cerevisiae catalyzes the oxidation of spermine and N(1)-acetylspermine to yield spermidine and 3-aminopropanal or N-acetyl-3-aminopropanal. The kinetic mechanism of the enzyme has been determined with both substrates. The initial velocity patterns are ping-pong, consistent with reduction being kinetically irreversible. Reduction of Fms1 by either substrate is biphasic. The rate constant for the rapid phase varies with the substrate concentration, with limiting rates for reduction of the enzyme of 126 and 1410 s(-1) and apparent K(d) values of 24.3 and 484 µM for spermine and N(1)-acetylspermine, respectively. The rapid phase is followed by a concentration-independent phase that is slower than turnover. The reaction of the reduced enzyme with oxygen is monophasic, with a rate constant of 402 mM(-1) s(-1) with spermine at 25 °C and 204 mM(-1) s(-1) with N(1)-acetylspermine at 4 °C and pH 9.0. This step is followed by rate-limiting product dissociation. The k(cat)/K(amine)-pH profiles are bell-shaped, with an average pK(a) value of 9.3 with spermine and pK(a) values of 8.3 and 9.6 with N(1)-acetylspermine. Both profiles are consistent with the active forms of substrates having two charged nitrogens. The pH profiles for the rate constant for flavin reduction show pK(a) values of 8.3 and 7.2 for spermine and N(1)-acetylspermine, respectively, for groups that must be unprotonated; these pK(a) values are assigned to the substrate N4. The k(cat)/K(O(2))-pH profiles show pK(a) values of 7.5 for spermine and 6.8 for N(1)-acetylspermine. With both substrates, the k(cat) value decreases when a single residue is protonated.

Mechanistic studies of human spermine oxidase: kinetic mechanism and pH effects.

In mammalian cells, the flavoprotein spermine oxidase (SMO) catalyzes the oxidation of spermine to spermidine and 3-aminopropanal. Mechanistic studies have been conducted with the recombinant human enzyme. The initial velocity pattern in which the ratio between the concentrations of spermine and oxygen is kept constant establishes the steady-state kinetic pattern as ping-pong. Reduction of SMO by spermine in the absence of oxygen is biphasic. The rate constant for the rapid phase varies with the substrate concentration, with a limiting value (k(3)) of 49 s(-1) and an apparent K(d) value of 48 microM at pH 8.3. The rate constant for the slow step is independent of the spermine concentration, with a value of 5.5 s(-1), comparable to the k(cat) value of 6.6 s(-1). The kinetics of the oxidative half-reaction depend on the aging time after the spermine and enzyme are mixed in a double-mixing experiment. At an aging time of 6 s, the reaction is monophasic with a second-order rate constant of 4.2 mM(-1) s(-1). At an aging time of 0.3 s, the reaction is biphasic with two second-order constants equal to 4.0 and 40 mM(-1) s(-1). Neither is equal to the k(cat)/K(O(2)) value of 13 mM(-1) s(-1). These results establish the existence of more than one pathway for the reaction of the reduced flavin intermediate with oxygen. The k(cat)/K(M) value for spermine exhibits a bell-shaped pH profile, with an average pK(a) value of 8.3. This profile is consistent with the active form of spermine having three charged nitrogens. The pH profile for k(3) shows a pK(a) value of 7.4 for a group that must be unprotonated. The pK(i)-pH profiles for the competitive inhibitors N,N'-dibenzylbutane-1,4-diamine and spermidine show that the fully protonated forms of the inhibitors and the unprotonated form of an amino acid residue with a pK(a) of approximately 7.4 in the active site are preferred for binding.