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MECHANISTIC STUDIES OF THE PLP-DEPENDENT ENZYME, METHIONINE GAMMA-LYASE AND THE HEME-DEPENDENT ENZYME, INDOLEAMINE 2,3-DIOXYGENASE

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Date Issued:
2021
Abstract/Description:
Methionine-γ-lyase (MGL) is a pyridoxal-5′-phosphate (PLP) dependent enzyme found in bacteria and protozoa that catalyzes a variety of reactions, including the γ-elimination of L-methionine (L-Met). Besides its physiological roles in organisms, MGL is also a therapeutic target for pathogenic diseases and cancer. Since MGL’s catalytic mechanism remains uncertain, a new spectrophotometric assay was validated for measuring the kinetics of MGL catalyzed reactions. Kinetic data and density functional theory (DFT) data for the γ-elimination reaction of L-Met and several other substrate analogues by MGL from P. gingivalis is reported. A direct correlation between experimental kcat values and DFT-calculated activation energies of the γ-cleavage reaction of the enamine intermediate was identified for various substrates. Considering these data, we propose a catalytic mechanism for MGL catalysis, whereby the γ-cleavage step is rate-limiting. This conclusion has direct implications for the rational design of substrates or inhibitors aimed at regulating MGL activity. PLP-dependent enzymes are present in all organisms and account for ~4% of all discovered enzyme activities. They all possess a common external aldimine and quinonoid intermediate at the start of their reaction pathway but likely diverge thereafter into different reaction pathways based on their substrate(s) and active site environment. To yield insight into the divergent reactivity of the quinonoid intermediate, DFT calculations of several PLP-dependent enzymes with unique reactivity revealed that the charge distribution is modulated on Cα and C4’, which allows for the regioselectivity of the quinonoid intermediate. The mammalian heme enzyme indoleamine 2,3-dioxygenase-1 (IDO1) catalyzes the first reaction of L-tryptophan (L-Trp) oxidation along the kynurenine pathway. IDO1 is a central immunoregulatory enzyme with important implications for inflammation, infectious disease, autoimmune disorders, and cancer. Since IDO1’s catalytic mechanism remains uncertain, kinetic data and DFT data for the dioxygenation reaction of L-Trp and several other substrate analogues by human IDO1 is reported. A direct correlation between experimental kcat values and DFT-calculated activation energies of the C2-O cleavage reaction of the L-Trp-epoxide intermediate was identified for various substrates. This conclusion has direct implications for the rational design of substrates or inhibitors aimed at regulating IDO1 activity and yields insight into heme-dependent dioxygenation chemistry.
Title: MECHANISTIC STUDIES OF THE PLP-DEPENDENT ENZYME, METHIONINE GAMMA-LYASE AND THE HEME-DEPENDENT ENZYME, INDOLEAMINE 2,3-DIOXYGENASE.
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Name(s): Foo, Timothy, author
Terentis, Andrew C. , Thesis advisor
Florida Atlantic University, Degree grantor
Department of Chemistry and Biochemistry
Charles E. Schmidt College of Science
Type of Resource: text
Genre: Electronic Thesis Or Dissertation
Date Created: 2021
Date Issued: 2021
Publisher: Florida Atlantic University
Place of Publication: Boca Raton, Fla.
Physical Form: application/pdf
Extent: 284 p.
Language(s): English
Abstract/Description: Methionine-γ-lyase (MGL) is a pyridoxal-5′-phosphate (PLP) dependent enzyme found in bacteria and protozoa that catalyzes a variety of reactions, including the γ-elimination of L-methionine (L-Met). Besides its physiological roles in organisms, MGL is also a therapeutic target for pathogenic diseases and cancer. Since MGL’s catalytic mechanism remains uncertain, a new spectrophotometric assay was validated for measuring the kinetics of MGL catalyzed reactions. Kinetic data and density functional theory (DFT) data for the γ-elimination reaction of L-Met and several other substrate analogues by MGL from P. gingivalis is reported. A direct correlation between experimental kcat values and DFT-calculated activation energies of the γ-cleavage reaction of the enamine intermediate was identified for various substrates. Considering these data, we propose a catalytic mechanism for MGL catalysis, whereby the γ-cleavage step is rate-limiting. This conclusion has direct implications for the rational design of substrates or inhibitors aimed at regulating MGL activity. PLP-dependent enzymes are present in all organisms and account for ~4% of all discovered enzyme activities. They all possess a common external aldimine and quinonoid intermediate at the start of their reaction pathway but likely diverge thereafter into different reaction pathways based on their substrate(s) and active site environment. To yield insight into the divergent reactivity of the quinonoid intermediate, DFT calculations of several PLP-dependent enzymes with unique reactivity revealed that the charge distribution is modulated on Cα and C4’, which allows for the regioselectivity of the quinonoid intermediate. The mammalian heme enzyme indoleamine 2,3-dioxygenase-1 (IDO1) catalyzes the first reaction of L-tryptophan (L-Trp) oxidation along the kynurenine pathway. IDO1 is a central immunoregulatory enzyme with important implications for inflammation, infectious disease, autoimmune disorders, and cancer. Since IDO1’s catalytic mechanism remains uncertain, kinetic data and DFT data for the dioxygenation reaction of L-Trp and several other substrate analogues by human IDO1 is reported. A direct correlation between experimental kcat values and DFT-calculated activation energies of the C2-O cleavage reaction of the L-Trp-epoxide intermediate was identified for various substrates. This conclusion has direct implications for the rational design of substrates or inhibitors aimed at regulating IDO1 activity and yields insight into heme-dependent dioxygenation chemistry.
Identifier: FA00013870 (IID)
Degree granted: Dissertation (Ph.D.)--Florida Atlantic University, 2021.
Collection: FAU Electronic Theses and Dissertations Collection
Note(s): Includes bibliography.
Subject(s): Lyases
Enzymes
Persistent Link to This Record: http://purl.flvc.org/fau/fd/FA00013870
Use and Reproduction: Copyright © is held by the author with permission granted to Florida Atlantic University to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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Host Institution: FAU
Is Part of Series: Florida Atlantic University Digital Library Collections.