Studies on the reaction mechanism of lactate oxidase. Formation of two covalent flavin-substrate adducts on reaction with glycollate.

Massey, Vincent; Ghisla, Sandro; Kieschke, Klaus

doi:10.1016/s0021-9258(19)85809-x

Cited by 45 publications

(5 citation statements)

References 26 publications

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“…A further difference between the two enzymes is in their reaction with glycolate. Lactate oxidase has been shown to be able to abstract either the re hydrogen or the si hydrogen from glycolate with the subsequent formation of the corresponding enantiomeric flavin N(5)-glycolyl adducts; the former results in normal catalysis with formate, carbon dioxide, and water as final products, but the latter results in the formation of a stable flavin N(5)-glycolyl adduct (Massey et al, 1980;. On the other hand, glycolate oxidase oxidizes glycolate to glyoxylate without any indication of the occurrence of such a catalytically incompetent adduct.…”

Section: Discussionmentioning

confidence: 99%

Expression of spinach glycolate oxidase in Saccharomyces cerevisiae: purification and characterization

Macheroux¹,

Massey²,

Thiele³

et al. 1991

Biochemistry

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Glycolate oxidase from spinach has been expressed in Saccharomyces cerevisiae. The active enzyme was purified to near-homogeneity (purification factor approximately 1400-fold) by means of hydroxyapatite and anion-exchange chromatography. The purified glycolate oxidase is nonfluorescent and has absorbance peaks at 448 (epsilon = 9200 M-1 cm-1) and 346 nm in 0.1 M phosphate buffer, pH 8.3. The large bathochromic shift of the near-UV band indicates that the N(3) position is deprotonated at pH 8.3. A pH titration revealed that the pK of the N(3) is shifted from 10.3 in free flavin to 6.4 in glycolate oxidase. Glycolate oxidase is competitively inhibited by oxalate with a Kd of 0.24 mM at 4 degrees C in 0.1 M phosphate buffer, pH 8.3. Three pieces of evidence demonstrate that glycolate oxidase stabilizes a negative charge at the N(1)-C(2 = O) locus: the enzyme forms a tight sulfite complex with a Kd of 2.7 x 10(-7) M and stabilizes the anionic flavosemiquinone and the benzoquinoid form of 8-mercapto-FMN. Steady-state analysis at pH 8.3, 4 degrees C, yielded a Km = 1 x 10(-3) M for glycolate and Km = 2.1 x 10(-4) M for oxygen. The turnover number has been determined to be 20 s-1. Stopped-flow studies of the reductive (k = 25 s-1) and oxidative (k = 8.5 x 10(4) M-1 s-1) half-reactions have identified the reduction of glycolate oxidase to be the rate-limiting step.

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Section: Discussionmentioning

confidence: 99%

Expression of spinach glycolate oxidase in Saccharomyces cerevisiae: purification and characterization

Macheroux¹,

Massey²,

Thiele³

et al. 1991

Biochemistry

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“…Although the enzyme is an internal monooxygenase which catalyzes the four-electron oxidation of L-lactate to acetate and carbon dioxide with the reduction of 02 to water, the mechanism is well established to proceed by oxidation of lactate to pyruvate, which is subsequently oxidatively decarboxylated (Lockridge et al, 1972). Recently, evidence of a covalent adduct of the substrate glycollate and N(5) of the flavin mononucleotide (FMN) coenzyme as a catalytically competent intermediate has been reported (Massey et al, 1980; Ghisla .…”

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confidence: 99%

Inactivation of L-lactate monooxygenase with 2,3-butanedione and phenylglyoxal

Peters¹,

Jones²,

Cromartie³

1981

Biochemistry

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L-Lactate monooxygenase from Mycobacterium phlei is inactivated by reaction either with 2,3-butanedione in borate or in 2,6-lutidine buffer or with phenyglyoxal in 2,6-lutidine buffer. The activation with 2.3 butanedione in borate buffer is irreversible in the presence of excess borate, but essentially complete recovery of activity occurs on exchange of phosphate for borate buffer. In 50 mM borate, inactivation with 2,3-butanedione exhibits saturation kinetics with respect to increasing concentrations of 2,3-butanedione, whereas second-order kinetics for inactivation are seen in 200 mM borate. In 2.6-lutidine buffer, the inactivation is rapid, irreversible on change of buffer, and second order overall. Complete inactivation of the enzyme by phenylglyoxal in 2,6-lutidine buffer occurs on incorporation of 2 equiv of phenylglyoxal per subunit, but only one arginyl residue per subunit is modified. The inactivation is irreversible and second order in phenyglyoxal. There is substantial protection from inactivation in the presence of D-lactate, a competitive inhibitor of the enzyme. It is suggested that an arginyl residue in the active site in L-lactate monooxygenase is involved in the binding of the carboxyl group of substrates to the enzyme. An explanation for the unusual kinetics of inactivation with 2,3-butanedione in borate and with phenylglyoxal in 2,6-lutidine is offered.

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“…This atom is required for the redox activity of the cofactor [ 193 ] and, hence, for the catalytic function of the enzyme. This conformation could not be achieved without the orientation promoted by the L171 and Y398 residues, which suggests that FA-26 could inhibit some enzymes with the same mechanism of action as MAO (type A) or other flavoenzymes as lactate oxidase [ 196 ]. According to these findings, FA-26 is predicted to act as a reversible or non-covalent MAO-B inhibitor as Safinamide or Moclobemide [ 175 , 197 ], which are recognized antidepressant drugs.…”

Section: Resultsmentioning

confidence: 99%

Rational Design of Multifunctional Ferulic Acid Derivatives Aimed for Alzheimer’s and Parkinson’s Diseases

et al. 2023

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Ferulic acid has numerous beneficial effects on human health, which are frequently attributed to its antioxidant behavior. In this report, many of them are reviewed, and 185 new ferulic acid derivatives are computationally designed using the CADMA-Chem protocol. Consequently, their chemical space was sampled and evaluated. To that purpose, selection and elimination scores were used, which are built from a set of descriptors accounting for ADME properties, toxicity, and synthetic accessibility. After the first screening, 12 derivatives were selected and further investigated. Their potential role as antioxidants was predicted from reactivity indexes directly related to the formal hydrogen atom transfer and the single electron transfer mechanisms. The best performing molecules were identified by comparisons with the parent molecule and two references: Trolox and α-tocopherol. Their potential as polygenic neuroprotectors was investigated through the interactions with enzymes directly related to the etiologies of Parkinson’s and Alzheimer’s diseases. These enzymes are acetylcholinesterase, catechol-O-methyltransferase, and monoamine oxidase B. Based on the obtained results, the most promising candidates (FA-26, FA-118, and FA-138) are proposed as multifunctional antioxidants with potential neuroprotective effects. The findings derived from this investigation are encouraging and might promote further investigations on these molecules.

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Studies on the reaction mechanism of lactate oxidase. Formation of two covalent flavin-substrate adducts on reaction with glycollate.

Cited by 45 publications

References 26 publications

Expression of spinach glycolate oxidase in Saccharomyces cerevisiae: purification and characterization

Expression of spinach glycolate oxidase in Saccharomyces cerevisiae: purification and characterization

Inactivation of L-lactate monooxygenase with 2,3-butanedione and phenylglyoxal

Rational Design of Multifunctional Ferulic Acid Derivatives Aimed for Alzheimer’s and Parkinson’s Diseases

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