N-linoleoylamino acids as chiral probes of substrate binding by soybean lipoxygenase-1

Charles H. Clapp, Justin Pachuski, Natasha F. Bassett, Kathleen A. Bishop, Gillian Carter, Megan Marie Young, Thomas Young, Yuhan Fu

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Lipoxygenases catalyze the oxygenation of polyunsaturated fatty acids and their derivatives to produce conjugated diene hydroperoxides. Soybean lipoxygenase-1 (SBLO-1) has been the subject of intensive structural and mechanistic study, but the manner in which this enzyme binds substrates is uncertain. Previous studies suggest that the fatty acyl group of the substrate binds in an internal cavity near the catalytic iron with the polar end at the surface of the protein or perhaps external to the protein. To test this model, we have investigated two pairs of enantiomeric N-linoleoylamino acids as substrates for SBLO-1. If the amino acid moiety binds external to the protein, the kinetics and product distribution should show little or no sensitivity to the stereochemical configuration of the amino acid moiety. Consistent with this expectation, N-linoleoyl-L-valine (LLV) and N-linoleoyl-D-valine (LDV) are both good substrates with kcat/Km values that are equal within error and about 40% higher than kcat/Km for linoleic acid. Experiments with N-linoleoyl-L-tryptophan (LLT) and N-linoleoyl-D-tryptophan (LDT) were complicated by the low critical micelle concentrations (CMC = 6–8 μM) of these substances. Below the CMC, LDT is a better substrate by a factor of 2.7. The rates of oxygenation of LDT and LLT continue to rise above the CMC, with modest stereoselectivity in favor of the D enantiomer. With all of the substrates tested, the major product is the 13(S)-hydroperoxide, and the distribution of minor products is not appreciably affected by the configuration of the amino acid moiety. The absence of stereoselectivity with LLV and LDV, the modest magnitude of the stereoselectivity with LLT and LDT, and the ability micellar forms of LLT and LDT to increase the concentration of available substrate are all consistent with the hypothesis that the amino acid moiety binds largely external to SBLO-1 and interacts with it only weakly.

Original languageEnglish (US)
Pages (from-to)170-177
Number of pages8
JournalBioorganic Chemistry
Volume78
DOIs
StatePublished - Aug 1 2018

Fingerprint

Tryptophan
Acids
Substrates
Valine
Stereoselectivity
Amino Acids
Oxygenation
Hydrogen Peroxide
Lipoxygenases
lipoxygenase L-1
Enantiomers
Critical micelle concentration
Micelles
Linoleic Acid
Unsaturated Fatty Acids
Membrane Proteins
Proteins
Iron
Derivatives
Kinetics

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Drug Discovery
  • Organic Chemistry

Cite this

Clapp, Charles H. ; Pachuski, Justin ; Bassett, Natasha F. ; Bishop, Kathleen A. ; Carter, Gillian ; Young, Megan Marie ; Young, Thomas ; Fu, Yuhan. / N-linoleoylamino acids as chiral probes of substrate binding by soybean lipoxygenase-1. In: Bioorganic Chemistry. 2018 ; Vol. 78. pp. 170-177.
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abstract = "Lipoxygenases catalyze the oxygenation of polyunsaturated fatty acids and their derivatives to produce conjugated diene hydroperoxides. Soybean lipoxygenase-1 (SBLO-1) has been the subject of intensive structural and mechanistic study, but the manner in which this enzyme binds substrates is uncertain. Previous studies suggest that the fatty acyl group of the substrate binds in an internal cavity near the catalytic iron with the polar end at the surface of the protein or perhaps external to the protein. To test this model, we have investigated two pairs of enantiomeric N-linoleoylamino acids as substrates for SBLO-1. If the amino acid moiety binds external to the protein, the kinetics and product distribution should show little or no sensitivity to the stereochemical configuration of the amino acid moiety. Consistent with this expectation, N-linoleoyl-L-valine (LLV) and N-linoleoyl-D-valine (LDV) are both good substrates with kcat/Km values that are equal within error and about 40{\%} higher than kcat/Km for linoleic acid. Experiments with N-linoleoyl-L-tryptophan (LLT) and N-linoleoyl-D-tryptophan (LDT) were complicated by the low critical micelle concentrations (CMC = 6–8 μM) of these substances. Below the CMC, LDT is a better substrate by a factor of 2.7. The rates of oxygenation of LDT and LLT continue to rise above the CMC, with modest stereoselectivity in favor of the D enantiomer. With all of the substrates tested, the major product is the 13(S)-hydroperoxide, and the distribution of minor products is not appreciably affected by the configuration of the amino acid moiety. The absence of stereoselectivity with LLV and LDV, the modest magnitude of the stereoselectivity with LLT and LDT, and the ability micellar forms of LLT and LDT to increase the concentration of available substrate are all consistent with the hypothesis that the amino acid moiety binds largely external to SBLO-1 and interacts with it only weakly.",
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N-linoleoylamino acids as chiral probes of substrate binding by soybean lipoxygenase-1. / Clapp, Charles H.; Pachuski, Justin; Bassett, Natasha F.; Bishop, Kathleen A.; Carter, Gillian; Young, Megan Marie; Young, Thomas; Fu, Yuhan.

In: Bioorganic Chemistry, Vol. 78, 01.08.2018, p. 170-177.

Research output: Contribution to journalArticle

TY - JOUR

T1 - N-linoleoylamino acids as chiral probes of substrate binding by soybean lipoxygenase-1

AU - Clapp, Charles H.

AU - Pachuski, Justin

AU - Bassett, Natasha F.

AU - Bishop, Kathleen A.

AU - Carter, Gillian

AU - Young, Megan Marie

AU - Young, Thomas

AU - Fu, Yuhan

PY - 2018/8/1

Y1 - 2018/8/1

N2 - Lipoxygenases catalyze the oxygenation of polyunsaturated fatty acids and their derivatives to produce conjugated diene hydroperoxides. Soybean lipoxygenase-1 (SBLO-1) has been the subject of intensive structural and mechanistic study, but the manner in which this enzyme binds substrates is uncertain. Previous studies suggest that the fatty acyl group of the substrate binds in an internal cavity near the catalytic iron with the polar end at the surface of the protein or perhaps external to the protein. To test this model, we have investigated two pairs of enantiomeric N-linoleoylamino acids as substrates for SBLO-1. If the amino acid moiety binds external to the protein, the kinetics and product distribution should show little or no sensitivity to the stereochemical configuration of the amino acid moiety. Consistent with this expectation, N-linoleoyl-L-valine (LLV) and N-linoleoyl-D-valine (LDV) are both good substrates with kcat/Km values that are equal within error and about 40% higher than kcat/Km for linoleic acid. Experiments with N-linoleoyl-L-tryptophan (LLT) and N-linoleoyl-D-tryptophan (LDT) were complicated by the low critical micelle concentrations (CMC = 6–8 μM) of these substances. Below the CMC, LDT is a better substrate by a factor of 2.7. The rates of oxygenation of LDT and LLT continue to rise above the CMC, with modest stereoselectivity in favor of the D enantiomer. With all of the substrates tested, the major product is the 13(S)-hydroperoxide, and the distribution of minor products is not appreciably affected by the configuration of the amino acid moiety. The absence of stereoselectivity with LLV and LDV, the modest magnitude of the stereoselectivity with LLT and LDT, and the ability micellar forms of LLT and LDT to increase the concentration of available substrate are all consistent with the hypothesis that the amino acid moiety binds largely external to SBLO-1 and interacts with it only weakly.

AB - Lipoxygenases catalyze the oxygenation of polyunsaturated fatty acids and their derivatives to produce conjugated diene hydroperoxides. Soybean lipoxygenase-1 (SBLO-1) has been the subject of intensive structural and mechanistic study, but the manner in which this enzyme binds substrates is uncertain. Previous studies suggest that the fatty acyl group of the substrate binds in an internal cavity near the catalytic iron with the polar end at the surface of the protein or perhaps external to the protein. To test this model, we have investigated two pairs of enantiomeric N-linoleoylamino acids as substrates for SBLO-1. If the amino acid moiety binds external to the protein, the kinetics and product distribution should show little or no sensitivity to the stereochemical configuration of the amino acid moiety. Consistent with this expectation, N-linoleoyl-L-valine (LLV) and N-linoleoyl-D-valine (LDV) are both good substrates with kcat/Km values that are equal within error and about 40% higher than kcat/Km for linoleic acid. Experiments with N-linoleoyl-L-tryptophan (LLT) and N-linoleoyl-D-tryptophan (LDT) were complicated by the low critical micelle concentrations (CMC = 6–8 μM) of these substances. Below the CMC, LDT is a better substrate by a factor of 2.7. The rates of oxygenation of LDT and LLT continue to rise above the CMC, with modest stereoselectivity in favor of the D enantiomer. With all of the substrates tested, the major product is the 13(S)-hydroperoxide, and the distribution of minor products is not appreciably affected by the configuration of the amino acid moiety. The absence of stereoselectivity with LLV and LDV, the modest magnitude of the stereoselectivity with LLT and LDT, and the ability micellar forms of LLT and LDT to increase the concentration of available substrate are all consistent with the hypothesis that the amino acid moiety binds largely external to SBLO-1 and interacts with it only weakly.

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