Oxygen and hydrogen isotope effects in an active site tyrosine to phenylalanine mutant of peptidylglycine α-hydroxylating monooxygenase: Mechanistic implications

Wilson A. Francisco, Ninian Blackburn, Judith P. Klinman

Research output: Contribution to journalArticle

57 Citations (Scopus)

Abstract

Peptidylglycine α-hydroxylating monooxygenase (PHM) and dopamine β-monooxygenase (DβM) are homologous copper-containing enzymes that catalyze an oxygen-dependent hydroxylation of peptide-extended glycine residues and phenethylamines, respectively. The mechanism whereby these enzymes activate molecular oxygen and the C-H bond of substrate has been the subject of numerous studies, and various mechanisms have been put forth. From the magnitude of 18O isotope effects as a function of substrate structure in DβM, an active site tyrosine had been proposed to function in the reductive activation of Cu(II)-OOH to generate a reactive copper-oxo species [Tian et al. (1994) Biochemistry 33, 226]. The presence of a tyrosine residue, Y318, in the active site of PHM was subsequently confirmed from crystallographic studies [Prigge et al. (1997) Science 278, 1300]. We now report extensive kinetic and isotope effect studies on the Y318F mutant form of PHM, analyzing the role of this tyrosine in the catalytic mechanism. It is found that the Y318F mutant has intrinsic hydrogen and 18O isotope effects that are within experimental error of the wild-type enzyme and that the mutation causes only a slight reduction in the rate constant for C-H bond cleavage. These findings, together with the recent demonstration that C-H activation in PHM is dominated by quantum mechanical tunneling [Francisco et al. (2002) J. Am. Chem. Soc. 124, 8194], necessitate a reexamination of plausible mechanisms for this unique class of copper enzymes.

Original languageEnglish (US)
Pages (from-to)1813-1819
Number of pages7
JournalBiochemistry
Volume42
Issue number7
DOIs
StatePublished - Feb 25 2003

Fingerprint

Oxygen Isotopes
Mixed Function Oxygenases
Phenylalanine
Isotopes
Tyrosine
Hydrogen
Catalytic Domain
Oxygen
Copper
Enzymes
Dopamine
Phenethylamines
Hydroxylation
Chemical activation
Biochemistry
Glycine
Molecular oxygen
Substrates
Peptides
Mutation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Oxygen and hydrogen isotope effects in an active site tyrosine to phenylalanine mutant of peptidylglycine α-hydroxylating monooxygenase : Mechanistic implications. / Francisco, Wilson A.; Blackburn, Ninian; Klinman, Judith P.

In: Biochemistry, Vol. 42, No. 7, 25.02.2003, p. 1813-1819.

Research output: Contribution to journalArticle

@article{91f3282d5c1c4e64ac348534134c4a27,
title = "Oxygen and hydrogen isotope effects in an active site tyrosine to phenylalanine mutant of peptidylglycine α-hydroxylating monooxygenase: Mechanistic implications",
abstract = "Peptidylglycine α-hydroxylating monooxygenase (PHM) and dopamine β-monooxygenase (DβM) are homologous copper-containing enzymes that catalyze an oxygen-dependent hydroxylation of peptide-extended glycine residues and phenethylamines, respectively. The mechanism whereby these enzymes activate molecular oxygen and the C-H bond of substrate has been the subject of numerous studies, and various mechanisms have been put forth. From the magnitude of 18O isotope effects as a function of substrate structure in DβM, an active site tyrosine had been proposed to function in the reductive activation of Cu(II)-OOH to generate a reactive copper-oxo species [Tian et al. (1994) Biochemistry 33, 226]. The presence of a tyrosine residue, Y318, in the active site of PHM was subsequently confirmed from crystallographic studies [Prigge et al. (1997) Science 278, 1300]. We now report extensive kinetic and isotope effect studies on the Y318F mutant form of PHM, analyzing the role of this tyrosine in the catalytic mechanism. It is found that the Y318F mutant has intrinsic hydrogen and 18O isotope effects that are within experimental error of the wild-type enzyme and that the mutation causes only a slight reduction in the rate constant for C-H bond cleavage. These findings, together with the recent demonstration that C-H activation in PHM is dominated by quantum mechanical tunneling [Francisco et al. (2002) J. Am. Chem. Soc. 124, 8194], necessitate a reexamination of plausible mechanisms for this unique class of copper enzymes.",
author = "Francisco, {Wilson A.} and Ninian Blackburn and Klinman, {Judith P.}",
year = "2003",
month = "2",
day = "25",
doi = "10.1021/bi020592t",
language = "English (US)",
volume = "42",
pages = "1813--1819",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "7",

}

TY - JOUR

T1 - Oxygen and hydrogen isotope effects in an active site tyrosine to phenylalanine mutant of peptidylglycine α-hydroxylating monooxygenase

T2 - Mechanistic implications

AU - Francisco, Wilson A.

AU - Blackburn, Ninian

AU - Klinman, Judith P.

PY - 2003/2/25

Y1 - 2003/2/25

N2 - Peptidylglycine α-hydroxylating monooxygenase (PHM) and dopamine β-monooxygenase (DβM) are homologous copper-containing enzymes that catalyze an oxygen-dependent hydroxylation of peptide-extended glycine residues and phenethylamines, respectively. The mechanism whereby these enzymes activate molecular oxygen and the C-H bond of substrate has been the subject of numerous studies, and various mechanisms have been put forth. From the magnitude of 18O isotope effects as a function of substrate structure in DβM, an active site tyrosine had been proposed to function in the reductive activation of Cu(II)-OOH to generate a reactive copper-oxo species [Tian et al. (1994) Biochemistry 33, 226]. The presence of a tyrosine residue, Y318, in the active site of PHM was subsequently confirmed from crystallographic studies [Prigge et al. (1997) Science 278, 1300]. We now report extensive kinetic and isotope effect studies on the Y318F mutant form of PHM, analyzing the role of this tyrosine in the catalytic mechanism. It is found that the Y318F mutant has intrinsic hydrogen and 18O isotope effects that are within experimental error of the wild-type enzyme and that the mutation causes only a slight reduction in the rate constant for C-H bond cleavage. These findings, together with the recent demonstration that C-H activation in PHM is dominated by quantum mechanical tunneling [Francisco et al. (2002) J. Am. Chem. Soc. 124, 8194], necessitate a reexamination of plausible mechanisms for this unique class of copper enzymes.

AB - Peptidylglycine α-hydroxylating monooxygenase (PHM) and dopamine β-monooxygenase (DβM) are homologous copper-containing enzymes that catalyze an oxygen-dependent hydroxylation of peptide-extended glycine residues and phenethylamines, respectively. The mechanism whereby these enzymes activate molecular oxygen and the C-H bond of substrate has been the subject of numerous studies, and various mechanisms have been put forth. From the magnitude of 18O isotope effects as a function of substrate structure in DβM, an active site tyrosine had been proposed to function in the reductive activation of Cu(II)-OOH to generate a reactive copper-oxo species [Tian et al. (1994) Biochemistry 33, 226]. The presence of a tyrosine residue, Y318, in the active site of PHM was subsequently confirmed from crystallographic studies [Prigge et al. (1997) Science 278, 1300]. We now report extensive kinetic and isotope effect studies on the Y318F mutant form of PHM, analyzing the role of this tyrosine in the catalytic mechanism. It is found that the Y318F mutant has intrinsic hydrogen and 18O isotope effects that are within experimental error of the wild-type enzyme and that the mutation causes only a slight reduction in the rate constant for C-H bond cleavage. These findings, together with the recent demonstration that C-H activation in PHM is dominated by quantum mechanical tunneling [Francisco et al. (2002) J. Am. Chem. Soc. 124, 8194], necessitate a reexamination of plausible mechanisms for this unique class of copper enzymes.

UR - http://www.scopus.com/inward/record.url?scp=0345270446&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0345270446&partnerID=8YFLogxK

U2 - 10.1021/bi020592t

DO - 10.1021/bi020592t

M3 - Article

C2 - 12590568

AN - SCOPUS:0345270446

VL - 42

SP - 1813

EP - 1819

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 7

ER -