Catalytic Mechanism of a Heme and Tyrosyl Radical-Containing Fatty Acid α-(Di)oxygenase

Mukherjee, Arnab; Angeles‐Boza, Alfredo M.; Huff, Gregory S.; Roth, Justine P.

doi:10.1021/ja104180v

Cited by 18 publications

(132 citation statements)

References 86 publications

(133 reference statements)

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“…This behavior is consistent with the observed deuterium and 18 O KIEs but raises questions concerning how their magnitudes might depend upon polarization of the hydrogen transfer in transition states with differently sized AA-and LAderived peroxyl radicals (27,35).…”

Section: Discussionsupporting

confidence: 81%

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A Revised Mechanism for Human Cyclooxygenase-2

Liu

Roth

2016

Journal of Biological Chemistry

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The mechanism of -6 polyunsaturated fatty acid oxidation by wild-type cyclooxygenase 2 and the Y334F variant, lacking a conserved hydrogen bond to the catalytic tyrosyl radical/tyrosine, was examined for the first time under physiologically relevant conditions. The enzymes show apparent bimolecular rate constants and deuterium kinetic isotope effects that increase in proportion to co-substrate concentrations before converging to limiting values. The trends exclude multiple dioxygenase mechanisms as well as the proposal that initial hydrogen atom abstraction from the fatty acid is the first irreversible step in catalysis. Temperature dependent kinetic studies reinforce the novel finding that hydrogen transfer from the reduced catalytic tyrosine to a terminal peroxyl radical is the first irreversible step that controls regio-and stereospecific product formation.Cyclooxygenases 1 and 2 (COX-1 and COX-2) 2 are tyrosyl radical-utilizing hemoproteins with dioxygenase and peroxidase activities (1-3). Also known as prostaglandin H synthases, these structurally homologous enzymes are expressed by distinct genes, resulting in differences in cellular localization and regulation. COX-1 and COX-2 use O 2 to oxidize arachidonic acid (AA) as well as linoleic acid (LA), its dietary precursor, by Equations 1 and 2. COX-1 has a smaller active site, resulting in greater specificity for AA over LA than seen in the larger, more promiscuous COX-2 (4).Each dioxygenase reaction starts with initial hydrogen abstraction (from AA or LA) by a catalytic tyrosyl radical. Antarafacial O 2 trapping (of AA ⅐ or LA ⅐ ) leads to a peroxyl radical that reoxidizes the catalytic tyrosine in a second hydrogen transfer step, thus propagating catalysis. The prostaglandin G 2 (PGG 2 ) and acyclic hydroperoxide compounds (HPETEs and HPODEs) formed are processed by the enzyme's peroxidase activity, which consumes two reducing equivalents to generate prostaglandin H 2 (PGH 2 ) and acyclic hydroxylated compounds (hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecadienoic acids (HODEs)) together with H 2 O. In vitro studies commonly use phenol as the reductant. Earlier work demonstrated simple, saturating behavior when phenol reacts with COX-2 (5) but not COX-1 where acceleration and inhibition of dioxygenase catalysis are observed upon raising the phenol concentration (6).AA and LA bind to COX-2 in similar L-shaped conformations (Fig. 1). The reactive bisallylic C-H bond is thereby positioned close to the catalytic tyrosyl radical (Tyr-371 ⅐ ) (7, 8). Tyr-371 ⅐ forms rapidly upon exposure of the enzyme containing Fe(III)(protoporphyrin IX (Por)) to hydroperoxide compounds present at trace levels in AA and LA preparations (9). A Fe(IV)ϭO(Por ϩ ⅐ ) intermediate, typical in heme peroxidases, has been implicated as the oxidant (10). This species resides approximately 5 Å away from Tyr-371, which hydrogen bonds to 12). By analogy, Mn(III)(Por)-reconstituted COX-2 likely reacts with hydroperoxide compounds via a Mn(V)ϭO(Por) intermediate to produce Tyr-371 ...

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Section: Discussionsupporting

confidence: 81%

“…Each increases to a limiting deuterium KIE at sufficiently high co-substrate concentration. The apparent competitive deuterium KIEs upon LA consumption were analyzed at varying [O 2 ] to test the kinetic mechanism and the relationship of (27,35).…”

Section: Discussionmentioning

confidence: 99%

A Revised Mechanism for Human Cyclooxygenase-2

Liu

Roth

2016

Journal of Biological Chemistry

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“…Despite the significant restrictions imposed by the extended inserts, two small channels exist on the surface of the enzyme that facilitate access of H 2 O 2 to the distal face of the heme. The restricted access may protect the enzyme from overoxidation when high concentrations of H 2 O 2 are present (35). …”

Section: Discussionmentioning

confidence: 99%

The Crystal Structure of α-Dioxygenase Provides Insight into Diversity in the Cyclooxygenase-Peroxidase Superfamily

et al. 2013

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α-Dioxygenases (α-DOX) oxygenate fatty acids into 2R-hydroperoxides. Despite low sequence identity, α-DOX share common catalytic features with Cyclooxygenases (COX), including the use of a tyrosyl radical during catalysis. We determined the x-ray crystal structure of Arabidopsis thaliana α-DOX to 1.5Å resolution. The α-DOX structure is monomeric, predominantly α-helical and comprised of two domains. The base domain exhibits low structural homology with the membrane-binding domain of COX but lies in a similar position with respect to the catalytic domain. The catalytic domain shows the highest similarity with the COX catalytic domain, where 21 of the 22 α-helical elements are conserved. Helices H2, H6, H8, and H17 form the heme binding cleft and walls of the active site channel. His-318, Thr-323, and Arg-566 are located near the catalytic tyrosine, Tyr-386, at the apex of the channel, where they interact with a chloride ion. Substitutions at these positions, coupled with kinetic analyses confirm previous hypotheses that implicate these residues as being involved in binding and orienting the carboxylate group of the fatty acid for optimal catalysis. Unique to α-DOX is the presence of two extended inserts on the surface of the enzyme that restrict access to the distal face of the heme, providing an explanation for the observed reduced peroxidase activity of the enzyme. The α-DOX structure represents the first member of the α-DOX subfamily to be structurally characterized within the cyclooxygenase-peroxidase family of heme-containing proteins.

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“…Interest in enzymes that use tyrosyl radicals (YO · ) in redox catalysis has spurred investigations of a wealth of small molecule and protein‐based models. Natural enzymes that use the YOH/YO · redox couple include photosystem II [3], ribonucleotide reductases [4], cytochrome c oxidase and related oxygen reductases [5], galactose oxidase [6], prostaglandin synthase [7], and a fatty acid oxygenase [8]. Furthermore, YOH oxidation products are implicated in disease states related to oxidative stress [9].…”

Section: Introductionmentioning

confidence: 99%

Redox properties of tyrosine and related molecules

2011

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Redox reactions of tyrosine play key roles in many biological processes, including water oxidation and DNA synthesis. We first review the redox properties of tyrosine (and other phenols) in small molecules and related polypeptides, then report work on (H20)/(Y48)-modified Pseudomonas aeruginosa azurin. The crystal structure of this protein (1.18 Å resolution) shows that H20 is strongly hydrogen bonded to Y48 (2.7–2.8 Å tyrosine-O to histidine-N distance). A firm conclusion is that proper tuning of the tyrosine potential by a proton-accepting base is critical for biological redox functions.

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Catalytic Mechanism of a Heme and Tyrosyl Radical-Containing Fatty Acid α-(Di)oxygenase

Cited by 18 publications

References 86 publications

A Revised Mechanism for Human Cyclooxygenase-2

A Revised Mechanism for Human Cyclooxygenase-2

The Crystal Structure of α-Dioxygenase Provides Insight into Diversity in the Cyclooxygenase-Peroxidase Superfamily

Redox properties of tyrosine and related molecules

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