Mechanism of the Flavoprotein d-6-Hydroxynicotine Oxidase: Substrate Specificity, pH and Solvent Isotope Effects, and Roles of Key Active-Site Residues.

The flavoprotein d-6-hydroxynicotine oxidase catalyzes an early step in the oxidation of ( R)-nicotine, the oxidation of a carbon-nitrogen bond in the pyrrolidine ring of ( R)-6-hydroxynicotine. The enzyme is a member of the vanillyl alcohol oxidase/ p-cresol methylhydroxylase family of flavoproteins. The effects of substrate modifications on the steady-state and rapid-reaction kinetic parameters are not consistent with the quinone-methide mechanism of p-cresol methylhydroxylase. There is no solvent isotope effect on the kcat/ Kamine value with either ( R)-6-hydroxynicotine or the slower substrate ( R)-6-hydroxynornicotine. The effect of pH on the rapid-reaction kinetic parameters establishes that only the neutral form of the substrate and the correctly protonated form of the enzyme bind. The active-site residues Lys348, Glu350, and Glu352 are all properly positioned for substrate binding. The K348M substitution has only a small effect on the kinetic parameters; the E350A and E350Q substitutions decrease the kcat/ Kamine value by ∼20- and ∼220-fold, respectively, and the E352Q substitution decreases this parameter ∼3800-fold. The kcat/ Kamine-pH profile is bell-shaped. The p Ka values in that profile are altered by replacement of ( R)-6-hydroxynicotine with ( R)-6-hydroxynornicotine as the substrate and by the substitutions for Glu350 and Glu352, although the profiles remain bell-shaped. The results are consistent with a network of hydrogen-bonded residues in the active site being involved in binding the neutral form of the amine substrate, followed by the transfer of a hydride from the amine to the flavin.

13C kinetic isotope effects on the reaction of a flavin amine oxidase determined from whole molecule isotope effects.

A large number of flavoproteins catalyze the oxidation of amines. Because of the importance of these enzymes in metabolism, their mechanisms have previously been studied using deuterium, nitrogen, and solvent isotope effects. While these results have been valuable for computational studies to distinguish among proposed mechanisms, a measure of the change at the reacting carbon has been lacking. We describe here the measurement of a 13C kinetic isotope effect for a representative amine oxidase, polyamine oxidase. The isotope effect was determined by analysis of the isotopic composition of the unlabeled substrate, N, N'-dibenzyl-1,4-diaminopropane, to obtain a pH-independent value of 1.025. The availability of a 13C isotope effect for flavoprotein-catalyzed amine oxidation provides the first measure of the change in bond order at the carbon involved in this carbon-hydrogen bond cleavage and will be of value to understanding the transition state structure for this class of enzymes.

Characterization of unstable products of flavin- and pterin-dependent enzymes by continuous-flow mass spectrometry.

Continuous-flow mass spectrometry (CFMS) was used to monitor the products formed during the initial 0.25-20 s of the reactions catalyzed by the flavoprotein N-acetylpolyamine oxidase (PAO) and the pterin-dependent enzymes phenylalanine hydroxylase (PheH) and tyrosine hydroxylase (TyrH). N,N'-Dibenzyl-1,4-diaminobutane (DBDB) is a substrate for PAO for which amine oxidation is rate-limiting. CFMS of the reaction showed formation of an initial imine due to oxidation of an exo-carbon-nitrogen bond. Nonenzymatic hydrolysis of the imine formed benzaldehyde and N-benzyl-1,4-diaminobutane; the subsequent oxidation by PAO of the latter to an additional imine could also be followed. Measurement of the deuterium kinetic isotope effect on DBDB oxidation by CFMS yielded a value of 7.6 ± 0.3, in good agreement with a value of 6.7 ± 0.6 from steady-state kinetic analyses. In the PheH reaction, the transient formation of the 4a-hydroxypterin product was readily detected; tandem mass spectrometry confirmed attachment of the oxygen to C(4a). With wild-type TyrH, the 4a-hydroxypterin was also the product. In contrast, no product other than a dihydropterin could be detected in the reaction of the mutant protein E332A TyrH.

Mechanistic studies of the role of a conserved histidine in a mammalian polyamine oxidase.

Polyamine oxidases are peroxisomal flavoproteins that catalyze the oxidation of an endo carbon nitrogen bond of N1-acetylspermine in the catabolism of polyamines. While no structure has been reported for a mammalian polyamine oxidase, sequence alignments of polyamine oxidizing flavoproteins identify a conserved histidine residue. Based on the structure of a yeast polyamine oxidase, Saccharomyces cerevisiae Fms1, this residue has been proposed to hydrogen bond to the reactive nitrogen in the polyamine substrate. The corresponding histidine in mouse polyamine oxidase, His64, has been mutated to glutamine, asparagine, and alanine to determine if this residue plays a similar role in the mammalian enzymes. The kinetics of the mutant enzymes were examined with N1-acetylspermine and the slow substrates spermine and N,N'-dibenzyl-1,4-diaminobutane. On average the mutations result in a decrease of ~15-fold in the rate constant for amine oxidation. Rapid-reaction kinetic analyses established that amine oxidation is rate-limiting with spermine as substrate for the wild-type and mutant enzymes and for the H64N enzyme with N1-acetylspermine as substrate. The k(cat)/K(O(2)) value was unaffected by the mutations with N1-acetylspermine as substrate, but decreased ~55-fold with the two slower substrates. The results are consistent with this residue assisting in properly positioning the amine substrate for oxidation.

Accumulation of tetrahedral intermediates in cholinesterase catalysis: a secondary isotope effect study.

In a previous communication, kinetic ß-deuterium secondary isotope effects were reported that support a mechanism for substrate-activated turnover of acetylthiocholine by human butyrylcholinesterase (BuChE) wherein the accumulating reactant state is a tetrahedral intermediate ( Tormos , J. R. ; et al. J. Am. Chem. Soc. 2005 , 127 , 14538 - 14539 ). In this contribution additional isotope effect experiments are described with acetyl-labeled acetylthiocholines (CL(3)COSCH(2)CH(2)N(+)Me(3); L = H or D) that also support accumulation of the tetrahedral intermediate in Drosophila melanogaster acetylcholinesterase (DmAChE) catalysis. In contrast to the aforementioned BuChE-catalyzed reaction, for this reaction the dependence of initial rates on substrate concentration is marked by pronounced substrate inhibition at high substrate concentrations. Moreover, kinetic ß-deuterium secondary isotope effects for turnover of acetylthiocholine depended on substrate concentration, and gave the following: (D3)k(cat)/K(m) = 0.95 ± 0.03, (D3)k(cat) = 1.12 ± 0.02 and (D3)ßk(cat) = 0.97 ± 0.04. The inverse isotope effect on k(cat)/K(m) is consistent with conversion of the sp(2)-hybridized substrate carbonyl in the E + A reactant state into a quasi-tetrahedral transition state in the acylation stage of catalysis, whereas the markedly normal isotope effect on k(cat) is consistent with hybridization change from sp(3) toward sp(2) as the reactant state for deacylation is converted into the subsequent transition state. Transition states for Drosophila melanogaster AChE-catalyzed hydrolysis of acetylthiocholine were further characterized by measuring solvent isotope effects and determining proton inventories. These experiments indicated that the transition state for rate-determining decomposition of the tetrahedral intermediate is stabilized by multiple protonic interactions. Finally, a simple model is proposed for the contribution that tetrahedral intermediate stabilization provides to the catalytic power of acetylcholinesterase.

Identification of a hypothetical protein from Podospora anserina as a nitroalkane oxidase.

The flavoprotein nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of primary and secondary nitroalkanes to their respective aldehydes and ketones. Structurally, the enzyme is a member of the acyl-CoA dehydrogenase superfamily. To date no enzymes other than that from F. oxysporum have been annotated as NAOs. To identify additional potential NAOs, the available database was searched for enzymes in which the active site residues Asp402, Arg409, and Ser276 were conserved. Of the several fungal enzymes identified in this fashion, PODANSg2158 from Podospora anserina was selected for expression and characterization. The recombinant enzyme is a flavoprotein with activity on nitroalkanes comparable to the F. oxysporum NAO, although the substrate specificity is somewhat different. Asp399, Arg406, and Ser273 in PODANSg2158 correspond to the active site triad in F. oxysporum NAO. The k(cat)/K(M)-pH profile with nitroethane shows a pK(a) of 5.9 that is assigned to Asp399 as the active site base. Mutation of Asp399 to asparagine decreases the k(cat)/K(M) value for nitroethane over 2 orders of magnitude. The R406K and S373A mutations decrease this kinetic parameter by 64- and 3-fold, respectively. The structure of PODANSg2158 has been determined at a resolution of 2.0 A, confirming its identification as an NAO.

A secondary isotope effect study of equine serum butyrylcholinesterase-catalyzed hydrolysis of acetylthiocholine.

beta-Secondary deuterium isotope effects have been measured for equine serum butyrylcholinesterase-catalyzed hydrolysis of acetyl-L(3)-thiocholine (L=H or (2)H). The dependencies of initial rates on isotopic substrate concentrations show close adherence to Michaelis-Menten kinetics, and yield the following isotope effects: (D3)k(cat)/K(m)=0.98+/-0.02 and (D3)k(cat)=1.10+/-0.02. The modestly inverse isotope effect on k(cat)/K(m) is consistent with partial rate limitation by a step that converts the sp(2)-hybridized ester carbonyl of the E+A reactant state into a quasi-tetrahedral transition state in the acylation stage of catalysis. On the other hand, the markedly normal isotope effect on k(cat) indicates that the Michaelis complex that accumulates at substrate saturation of the active site during catalytic turnover is a tetrahedral intermediate, whose decomposition is the rate-limiting step. These results compliment a previous report [J.R. Tormos et al., J. Am. Chem. Soc. 127 (2005) 14538-14539] that showed that substrate-activated hydrolysis of acetylthiocholine (ATCh), catalyzed by recombinant human butyrylcholinesterase, is also rate limited by decomposition of an accumulating tetrahedral intermediate.

The reactant state for substrate-activated turnover of acetylthiocholine by butyrylcholinesterase is a tetrahedral intermediate.

Secondary beta-deuterium kinetic isotope effects have been measured as a function of substrate concentration for recombinant human butyrylcholinesterase-catalyzed hydrolysis of acetyl-L3-thiocholine (L = 1H or 2H). The isotope effect on V/K is inverse, D3V/K = 0.93 +/- 0.03, which is consistent with conversion of the sp2 hybridized carbonyl carbon of the scissile ester bond of the E + A reactant state to a quasi-tetrahedral structure in the acylation transition state. In contrast, the isotope effect on Vmax under conditions of substrate activation is markedly normal, D3(betaVmax) = 1.29 +/- 0.06, an observation that is consistent with accumulation of a tetrahedral intermediate as the reactant state for catalytic turnover. Generally, tetrahedral intermediates for nonenzymatic ester hydrolyses are high-energy steady-state intermediates. Apparently, butyrylcholinesterase displays an unusual ability to stabilize such intermediates. Hence, the catalytic power of cholinesterases can largely be understood in terms of their ability to stabilize tetrahedral intermediates in the multistep reaction mechanism.