Nitrogen Isotope Effects As Probes of the Mechanism of <scp>d</scp>-Amino Acid Oxidase

Kurtz, Kevin A.; Rishavy, Mark A.; Cleland, W. W.; Fitzpatrick, Paul F.

doi:10.1021/ja002528+

Cited by 69 publications

(71 citation statements)

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“…Oxidation of L-alanine by TMO has an isotope effect of 1.0145 ± 0.0007 at pH 8. This value is in agreement with the value of 1.013 previously measured for D-serine oxidation by D-amino acid oxidase at pH 7.5 (35), further suggesting that both enzymes utilize a similar chemical mechanism.…”

Section: Discussionsupporting

confidence: 92%

Mechanistic Studies of the Flavoenzyme Tryptophan 2-Monooxygenase: Deuterium and ¹⁵N Kinetic Isotope Effects on Alanine Oxidation by an l-Amino Acid Oxidase

et al. 2006

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Tryptophan 2-monooxygenase (TMO) from Pseudomonas savastanoi catalyzes the oxidative decarboxylation of L-tryptophan during the biosynthesis of indoleacetic acid. Structurally and mechanistically the enzyme is a member of the family of L-amino acid oxidases. Deuterium and 15 N kinetic isotope effects were used to probe the chemical mechanism of L-alanine oxidation by TMO. The primary deuterium kinetic isotope effect was pH-independent over the pH range 6.5-10, with an average value of 6.0 ± 0.5, consistent with this being the intrinsic value. The deuterium isotope effect on the rate constant for flavin reduction by alanine was 6.3 ± 0.9; no intermediate flavin species were observed during flavin reduction. The 15 V/K alanine value was 1.0145 ± 0.0007 at pH 8. NMR analyses give an equilibrium 15 N isotope effect for deprotonation of the alanine amino group of 1.0233 ± 0.0004, allowing calculation of the 15 N isotope effect on the CH bond cleavage step of 0.9917 ± 0.0006. The results are consistent with TMO oxidation of alanine occurring through a hydride transfer mechanism.The flavoenzyme tryptophan 2-monooxygenase (TMO1) from Pseudomonas savastanoi catalyzes the oxidative decarboxylation of L-tryptophan (Scheme 1) in the first step of a twostep biosynthetic pathway for the plant hormone indoleacetic acid (10-12). The kinetic mechanism of TMO has been determined with its fastest substrate L-tryptophan (13), and can be divided into two half-reactions (Scheme 2). The reductive half-reaction involves cleavage of the α-CH bond of the amino acid (AA) and transfer of a hydride equivalent to the FAD to form the enzyme-bound imino acid. This is identical to the reaction of the flavoprotein L-amino acid oxidases and similar to the general reaction of flavoprotein amine oxidases. In the oxidative half-reaction of TMO, the reduced cofactor reacts with oxygen to produce hydrogen peroxide (14). Decarboxylation of the imine acid to the amide is thought to occur through the reaction of the hydrogen peroxide with the imino acid still bound to the enzyme (pathway a), analogously to the mechanism proposed by Lockridge et al. (15) for the decarboxylation of pyruvate by lactate oxidase. Although indoleacetamide is the only product of tryptophan turnover by wild-type TMO, amino acid oxidation can be uncoupled from decarboxylation to yield a keto acid in mutant enzymes (pathway b) (16,17).Despite their ubiquity and functional diversity, all flavin-dependent amine oxidases have thus far fallen into two structural families. One family includes D-amino acid oxidase (18), monomeric sarcosine oxidase (19) and glycine oxidase (20), while monoamine oxidase (21), *Address correspondence to:Paul F. Fitzpatrick,.Department of Biochemistry and Biophysics, 2128 TAMU, College Station, TX 77843-2128, Ph: 979-845-5487, Fax: 979-845-4946, Email: fitzpat@tamu.edu † This work was supported by NIH grants R01 GM58698 (PFF), R01 GM18938 (WWC), and T32 GM08523 (ECR) NIH Public Access (22), and L-amino acid oxidase (23) represent a separate f...

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Section: Discussionsupporting

confidence: 92%

Mechanistic Studies of the Flavoenzyme Tryptophan 2-Monooxygenase: Deuterium and ¹⁵N Kinetic Isotope Effects on Alanine Oxidation by an l-Amino Acid Oxidase

et al. 2006

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“…The oxidation of d-serine was allowed to proceed till 40-60% completion before isolating the remaining amino acid and analyzing its nitrogen isotopic composition by isotope ratio mass spectrometry. 23 The 15 (k cat /K ser ) value was 1.0177 AE 0.007 at pH 7.5, decreasing to 0.9944 AE 0.0025 at pH 10.6. This pH-dependence is due to the 15 K eq effect on the amine pK a .…”

Section: Amine and Amino Acid Oxidasesmentioning

confidence: 93%

“…A remarkable diversity of mechanisms have been proposed for amine oxidation by flavoproteins. These include direct hydride transfer from the neutral amine to the flavin (Scheme 1), 12 nucleophilic addition of the amine to the flavin (Scheme 2), 13 single electron transfer reactions such as that in Scheme 3, 14 and formation of a substrate carbanion by loss of a proton (Scheme 4). 15 The paradigm for studies of flavoprotein amine oxidases has been DAAO.…”

Section: Amine and Amino Acid Oxidasesmentioning

confidence: 99%

“…20,21 With this substrate, D (k cat /K ser ) has a pH-independent value of 4.5, identical to the isotope effect on the limiting rate of flavin reduction measured in a stopped-flow spectrophotomer, and D (k cat /K ser ) 1 15 N kinetic isotope effects were used to distinguish between a mechanism involving loss of this atom as a proton (Scheme 4) and a direct hydride transfer (Scheme 1). 12 Only in the case of the latter does rehybridization of nitrogen occur in the transition state for CH bond cleavage. The oxidation of d-serine was allowed to proceed till 40-60% completion before isolating the remaining amino acid and analyzing its nitrogen isotopic composition by isotope ratio mass spectrometry.…”

Section: Amine and Amino Acid Oxidasesmentioning

confidence: 99%

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Insights into the mechanisms of flavoprotein oxidases from kinetic isotope effects

Fitzpatrick

2007

Labelled Comp Radiopharmac

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Deuterium, solvent, and (15)N kinetic isotope effects have been used to probe the mechanisms by which flavoproteins oxidize carbon-oxygen and carbon-nitrogen bonds in amines, hydroxy acids, and alcohols. For the amine oxidases d-amino acid oxidase, N-methyltryptophan oxidase, and tryptophan monooxygenase, d-serine, sarcosine, and alanine are slow substrates for which CH bond cleavage is fully rate limiting. Inverse isotope effects for each of 0.992-0.996 are consistent with a common mechanism involving hydride transfer from the uncharged amine. Computational analyses of possible mechanisms support this conclusion. Deuterium and solvent isotope effects with wild-type and mutant variants of the lactate dehydrogenase flavocytochrome b(2) show that OH and CH bond cleavage are not concerted, but become so in the Y254F enzyme. This is consistent with a highly asynchronous reaction in which OH bond cleavage precedes hydride transfer. The results of Hammett analyses and solvent and deuterium isotope effects support a similar mechanism for alcohol oxidase.

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“…the growing evidence that flavoproteins that oxidize carbon-nitrogen and carbon-oxygen bonds typically catalyze loss of the substrate hydrogen as a hydride (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). As is generally the case with flavoproteins, the catalytic mechanism of NAO can be divided into oxidative and reductive half reactions (13,14).…”

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Mechanistic and Structural Analyses of the Roles of Arg409 and Asp402 in the Reaction of the Flavoprotein Nitroalkane Oxidase^,

et al. 2007

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The flavoprotein nitroalkane oxidase (NAO) catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones. The enzyme is a homolog of acyl-CoA dehydrogenase. Asp402 in NAO has been proposed to be the active site base responsible for removing the substrate proton in the first catalytic step; structurally it corresponds to the glutamate which acts as the base in medium chain acyl-CoA dehydrogenase. In the active site of NAO, the carboxylate of Asp402 forms an ionic interaction with the side chain of Arg409. The R409K enzyme has now been characterized kinetically and structurally. The mutation results in a decrease in the rate constant for proton abstraction of 100-fold. Analysis of the three-dimensional structure of the R409K enzyme, determined by X-ray crystallography to a resolution of 2.65 Å, shows that the critical structural change is an increase in the distance between the carboxylate of Asp402 and the positively-charged nitrogen in the side chain of the residue at position 409. The D402E mutation results in a smaller decrease in the rate constant for proton abstraction of 18-fold. The structure of the D402E enzyme, determined at 2.4 Å resolution, shows that there is a smaller increase in the distance between Arg409 and the carboxylate at position 402, and the interaction of this residue with Ser276 is perturbed. These results establish the critical importance of the interaction between Asp402 and Arg409 for proton abstraction by nitroalkane oxidase.The flavoenzyme nitroalkane oxidase (NAO) catalyzes the oxidation of nitroalkanes to the corresponding aldehydes or ketones with consumption of molecular oxygen and release of nitrite and hydrogen peroxide (Scheme 1) (1). NAO is unusual in that it catalyzes substrate oxidation by removing a substrate proton to form a carbanion intermediate (1,2), in contrast to † This research was supported in part by grants to PFF from the NIH (GM58698) and The Welch Foundation (A-1245) and to AMO from the American Heart Association (Grant in Aid 0555286B), the Offices of Biological and Environmental Research of the US Department of Energy, and the NIH (2 P41 RR012408). Use of the National Synchrotron Light Source at Brookhaven National Laboratory was supported by the U.S. Department of Energy Office of Basic Energy Sciences, under Contract DE-AC02-98CH10886. ‡ The atomic coordinates and structure factors for NAO D402E and R409K have been deposited with the Protein Data Bank with the corresponding file names 2REH, 2rehsf, 2ZAF and rcsb027723, respectively. *Corresponding authors. PFF: phone, fax,; e-mail, fitzpat@tamu.edu. AMO: phone 631-344-4739; e-mail: amorv@bnl.gov. 1 Abbreviations: NAO, nitroalkane oxidase; ACAD, acyl-CoA dehydrogenase; K ne , the K m value for nitroethane as the substrate 2 For comparison, we examined Lys359, which is near Lys409 but is part of helix I. The Lys359 N□ atom forms ionic interactions with the backbone carbonyl oxygen atoms of residues Tyr398, Phe401, Gly403 and the OD1 atom of Asn405. The elect...

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Nitrogen Isotope Effects As Probes of the Mechanism of d-Amino Acid Oxidase

Cited by 69 publications

References 10 publications

Mechanistic Studies of the Flavoenzyme Tryptophan 2-Monooxygenase: Deuterium and ¹⁵N Kinetic Isotope Effects on Alanine Oxidation by an l-Amino Acid Oxidase

Mechanistic Studies of the Flavoenzyme Tryptophan 2-Monooxygenase: Deuterium and ¹⁵N Kinetic Isotope Effects on Alanine Oxidation by an l-Amino Acid Oxidase

Insights into the mechanisms of flavoprotein oxidases from kinetic isotope effects

Mechanistic and Structural Analyses of the Roles of Arg409 and Asp402 in the Reaction of the Flavoprotein Nitroalkane Oxidase^,

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Nitrogen Isotope Effects As Probes of the Mechanism of d-Amino Acid Oxidase

Cited by 69 publications

References 10 publications

Mechanistic Studies of the Flavoenzyme Tryptophan 2-Monooxygenase: Deuterium and 15N Kinetic Isotope Effects on Alanine Oxidation by an l-Amino Acid Oxidase

Mechanistic Studies of the Flavoenzyme Tryptophan 2-Monooxygenase: Deuterium and 15N Kinetic Isotope Effects on Alanine Oxidation by an l-Amino Acid Oxidase

Insights into the mechanisms of flavoprotein oxidases from kinetic isotope effects

Mechanistic and Structural Analyses of the Roles of Arg409 and Asp402 in the Reaction of the Flavoprotein Nitroalkane Oxidase,

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Mechanistic Studies of the Flavoenzyme Tryptophan 2-Monooxygenase: Deuterium and ¹⁵N Kinetic Isotope Effects on Alanine Oxidation by an l-Amino Acid Oxidase

Mechanistic Studies of the Flavoenzyme Tryptophan 2-Monooxygenase: Deuterium and ¹⁵N Kinetic Isotope Effects on Alanine Oxidation by an l-Amino Acid Oxidase

Mechanistic and Structural Analyses of the Roles of Arg409 and Asp402 in the Reaction of the Flavoprotein Nitroalkane Oxidase^,