Mutation of surface cysteine 374 to alanine in monoamine oxidase A alters substrate turnover and inactivation by cyclopropylamines

Ana Paula B. Vintém; Nigel T. Price; Richard B. Silverman; Rona R. Ramsay

doi:10.1016/j.bmc.2005.02.061

Mutation of surface cysteine 374 to alanine in monoamine oxidase A alters substrate turnover and inactivation by cyclopropylamines

Ana Paula B. Vintém, Nigel T. Price, Richard B. Silverman, Rona R. Ramsay^*

^*Corresponding author for this work

Chemistry

Research output: Contribution to journal › Article › peer-review

48 Scopus citations

Abstract

Modification of cysteine (Cys) residues inactivates monoamine oxidases (MAO) yet the crystal structure shows no conserved cysteines in the active site of MAO A (Ma, J. et al. J. Mol. Biol. 2004, 338, 103-114). MAO A cysteine 374 was mutated to alanine and the purified enzyme characterized kinetically. The mutant was active but had decreased k_cat/K_m values compared to the wild-type enzyme. Cyclopropylamine-containing mechanism-based inactivators similarly showed lower turnover rates. Spectral studies and measurement of free thiols established that 1-phenylcyclopropylamine (1-PCPA) formed an irreversible flavin adduct whereas 2-phenylcyclopropylamine (2-PCPA) and N-cyclo-α-methylbenzylamine (N-CαMBA) formed adducts that allowed reoxidation of the flavin on denaturation and decreased cysteine in both wild-type and mutant MAO A. In the 1-PCPA and N-CαMBA inactivations, the partition ratio was decreased by more than 50% in the mutant. The data suggest that mutation of Cys374 influences MAO A catalysis, which has implications for MAO susceptibility to redox damage. These results are compared with previous work on the equivalent residue in MAO B, namely, cysteine 365.

Original language	English (US)
Pages (from-to)	3487-3495
Number of pages	9
Journal	Bioorganic and Medicinal Chemistry
Volume	13
Issue number	10
DOIs	https://doi.org/10.1016/j.bmc.2005.02.061
State	Published - May 15 2005

Funding

This work was supported by the Scottish Executive Environment and Rural Affairs Department (to N.T.P.), by the National Institutes of Health (GM32634 to R.B.S.), and by AstraZeneca UK Ltd (to R.R.R.). A.P.B.V. thanks the Fundação para a Ciência e Tecnologia for her PhD studentship and the William Ramsay Henderson Trust for travel support. The technical assistance of Mrs Joan Riddell is gratefully acknowledged. We thank Dr. P. Urban, CNRS, France for the initial gift of the cDNA for MAO.

Keywords

Allosteric effect
Chemical mechanism
Cyclopropylamine
Cysteine modification
Monoamine oxidase

ASJC Scopus subject areas

Drug Discovery
Molecular Medicine
Molecular Biology
Biochemistry
Clinical Biochemistry
Pharmaceutical Science
Organic Chemistry

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10.1016/j.bmc.2005.02.061

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@article{67aef6164fa24561b4a3a1483fff0aca,

title = "Mutation of surface cysteine 374 to alanine in monoamine oxidase A alters substrate turnover and inactivation by cyclopropylamines",

abstract = "Modification of cysteine (Cys) residues inactivates monoamine oxidases (MAO) yet the crystal structure shows no conserved cysteines in the active site of MAO A (Ma, J. et al. J. Mol. Biol. 2004, 338, 103-114). MAO A cysteine 374 was mutated to alanine and the purified enzyme characterized kinetically. The mutant was active but had decreased kcat/Km values compared to the wild-type enzyme. Cyclopropylamine-containing mechanism-based inactivators similarly showed lower turnover rates. Spectral studies and measurement of free thiols established that 1-phenylcyclopropylamine (1-PCPA) formed an irreversible flavin adduct whereas 2-phenylcyclopropylamine (2-PCPA) and N-cyclo-α-methylbenzylamine (N-CαMBA) formed adducts that allowed reoxidation of the flavin on denaturation and decreased cysteine in both wild-type and mutant MAO A. In the 1-PCPA and N-CαMBA inactivations, the partition ratio was decreased by more than 50% in the mutant. The data suggest that mutation of Cys374 influences MAO A catalysis, which has implications for MAO susceptibility to redox damage. These results are compared with previous work on the equivalent residue in MAO B, namely, cysteine 365.",

keywords = "Allosteric effect, Chemical mechanism, Cyclopropylamine, Cysteine modification, Monoamine oxidase",

author = "Vint{\'e}m, {Ana Paula B.} and Price, {Nigel T.} and Silverman, {Richard B.} and Ramsay, {Rona R.}",

note = "Funding Information: This work was supported by the Scottish Executive Environment and Rural Affairs Department (to N.T.P.), by the National Institutes of Health (GM32634 to R.B.S.), and by AstraZeneca UK Ltd (to R.R.R.). A.P.B.V. thanks the Funda{\c c}{\~a}o para a Ci{\^e}ncia e Tecnologia for her PhD studentship and the William Ramsay Henderson Trust for travel support. The technical assistance of Mrs Joan Riddell is gratefully acknowledged. We thank Dr. P. Urban, CNRS, France for the initial gift of the cDNA for MAO.",

year = "2005",

month = may,

day = "15",

doi = "10.1016/j.bmc.2005.02.061",

language = "English (US)",

volume = "13",

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T1 - Mutation of surface cysteine 374 to alanine in monoamine oxidase A alters substrate turnover and inactivation by cyclopropylamines

AU - Vintém, Ana Paula B.

AU - Price, Nigel T.

AU - Silverman, Richard B.

AU - Ramsay, Rona R.

N1 - Funding Information: This work was supported by the Scottish Executive Environment and Rural Affairs Department (to N.T.P.), by the National Institutes of Health (GM32634 to R.B.S.), and by AstraZeneca UK Ltd (to R.R.R.). A.P.B.V. thanks the Fundação para a Ciência e Tecnologia for her PhD studentship and the William Ramsay Henderson Trust for travel support. The technical assistance of Mrs Joan Riddell is gratefully acknowledged. We thank Dr. P. Urban, CNRS, France for the initial gift of the cDNA for MAO.

PY - 2005/5/15

Y1 - 2005/5/15

N2 - Modification of cysteine (Cys) residues inactivates monoamine oxidases (MAO) yet the crystal structure shows no conserved cysteines in the active site of MAO A (Ma, J. et al. J. Mol. Biol. 2004, 338, 103-114). MAO A cysteine 374 was mutated to alanine and the purified enzyme characterized kinetically. The mutant was active but had decreased kcat/Km values compared to the wild-type enzyme. Cyclopropylamine-containing mechanism-based inactivators similarly showed lower turnover rates. Spectral studies and measurement of free thiols established that 1-phenylcyclopropylamine (1-PCPA) formed an irreversible flavin adduct whereas 2-phenylcyclopropylamine (2-PCPA) and N-cyclo-α-methylbenzylamine (N-CαMBA) formed adducts that allowed reoxidation of the flavin on denaturation and decreased cysteine in both wild-type and mutant MAO A. In the 1-PCPA and N-CαMBA inactivations, the partition ratio was decreased by more than 50% in the mutant. The data suggest that mutation of Cys374 influences MAO A catalysis, which has implications for MAO susceptibility to redox damage. These results are compared with previous work on the equivalent residue in MAO B, namely, cysteine 365.

AB - Modification of cysteine (Cys) residues inactivates monoamine oxidases (MAO) yet the crystal structure shows no conserved cysteines in the active site of MAO A (Ma, J. et al. J. Mol. Biol. 2004, 338, 103-114). MAO A cysteine 374 was mutated to alanine and the purified enzyme characterized kinetically. The mutant was active but had decreased kcat/Km values compared to the wild-type enzyme. Cyclopropylamine-containing mechanism-based inactivators similarly showed lower turnover rates. Spectral studies and measurement of free thiols established that 1-phenylcyclopropylamine (1-PCPA) formed an irreversible flavin adduct whereas 2-phenylcyclopropylamine (2-PCPA) and N-cyclo-α-methylbenzylamine (N-CαMBA) formed adducts that allowed reoxidation of the flavin on denaturation and decreased cysteine in both wild-type and mutant MAO A. In the 1-PCPA and N-CαMBA inactivations, the partition ratio was decreased by more than 50% in the mutant. The data suggest that mutation of Cys374 influences MAO A catalysis, which has implications for MAO susceptibility to redox damage. These results are compared with previous work on the equivalent residue in MAO B, namely, cysteine 365.

KW - Allosteric effect

KW - Chemical mechanism

KW - Cyclopropylamine

KW - Cysteine modification

KW - Monoamine oxidase

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SN - 0968-0896

VL - 13

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EP - 3495

JO - Bioorganic and Medicinal Chemistry

JF - Bioorganic and Medicinal Chemistry

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ER -

Mutation of surface cysteine 374 to alanine in monoamine oxidase A alters substrate turnover and inactivation by cyclopropylamines

Abstract

Funding

Keywords

ASJC Scopus subject areas

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