title: A copper-methionine interaction controls the pH-dependent activation of peptidylglycine monooxygenase.
abstract: The pH dependence of native peptidylglycine monooxygenase (PHM) and its M314H variant has been studied in detail. For wild-type (WT) PHM, the intensity of the Cu-S interaction visible in the Cu(I) extended X-ray absorption fine structure (EXAFS) data is inversely proportional to catalytic activity over the pH range of 3-8. A previous model based on more limited data was interpreted in terms of two protein conformations involving an inactive Met-on form and an active flexible Met-off form [Bauman, A. T., et al. (2006) Biochemistry 45, 11140-11150] that derived its catalytic activity from the ability to couple into vibrational modes critical for proton tunneling. The new studies comparing the WT and M314H variant have led to the evolution of this model, in which the Met-on form has been found to be derived from coordination of an additional Met residue, rather than a more rigid conformer of M314 as previously proposed. The catalytic activity of the mutant decreased by 96% because of effects on both k(cat) and K(M), but it displayed the same activity-pH profile with a maximum around pH 6. At pH 8, the reduced Cu(I) form gave spectra that could be simulated by replacement of the Cu(M) Cu-S(Met) interaction with a Cu-N/O interaction, but the data did not unambiguously assign the ligand to the imidazole side chain of H314. At pH 3.5, the EXAFS still showed the presence of a strong Cu-S interaction, establishing that the Met-on form observed at low pH in WT cannot be due to a strengthening of the Cu(M)-methionine interaction but must arise from a different Cu-S interaction. Therefore, lowering the pH causes a conformational change at one of the Cu centers that brings a new S donor residue into a favorable orientation for coordination to copper and generates an inactive form. Cys coordination is unlikely because all Cys residues in PHM are engaged in disulfide cross-links. Sequence comparison with the PHM homologues tyramine β-monooxygenase and dopamine β-monooxygenase suggests that M109 (adjacent to H site ligands H107 and H108) is the most likely candidate. A model is presented in which H108 is protonated with a pK(a) of 4.6 to generate the inactive low-pH form with Cu(H) coordinated by M109, H107, and H172.
authors.affiliation: Institute of Environmental Health, Oregon Health and Sciences University, Beaverton, Oregon 97006, United States.
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Bauman
authors.forename: Brenda A
authors.initials: BA
authors.lastname: Broers
authors.forename: Chelsey D
authors.initials: CD
authors.lastname: Kline
authors.forename: Ninian J
authors.initials: NJ
authors.lastname: Blackburn
mesh_terms: D019943:Amino Acid Substitution; D055162:Biocatalysis; D020134:Catalytic Domain; D003300:Copper / Q000737:chemistry*; D004789:Enzyme Activation; D005583:Fourier Analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008715:Methionine / Q000737:chemistry*; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D056928:X-Ray Absorption Spectroscopy
chemical_list: D009097:Multienzyme Complexes; D050505:Mutant Proteins; D011994:Recombinant Proteins; D003300:Copper; D008715:Methionine; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase
references.citation: J Am Chem Soc. 2010 Nov 10;132(44):15565-72
references.pmid: 20958070
references.citation: J Biol Chem. 2006 Feb 10;281(6):3013-6
references.pmid: 16301310
references.citation: Acc Chem Res. 2002 Mar;35(3):183-91
references.pmid: 11900522
references.citation: Protein Expr Purif. 2007 Jan;51(1):34-8
references.pmid: 16931045
references.citation: Proteins. 1994 Aug;19(4):302-9
references.pmid: 7984626
references.citation: Biochem J. 1984 May 1;219(3):985-90
references.pmid: 6743256
references.citation: J Biol Chem. 2001 Aug 31;276(35):33027-35
references.pmid: 11402049
references.citation: J Am Chem Soc. 2010 Mar 24;132(11):3770-7
references.pmid: 20192263
references.citation: J Biol Inorg Chem. 2010 Nov;15(8):1195-207
references.pmid: 20544364
references.citation: Science. 1997 Nov 14;278(5341):1300-5
references.pmid: 9360928
references.citation: J Am Chem Soc. 2008 Sep 10;130(36):11939-44
references.pmid: 18710228
references.citation: J Biol Chem. 2004 Nov 12;279(46):48159-67
references.pmid: 15337741
references.citation: J Biol Chem. 1981 Apr 10;256(7):3385-93
references.pmid: 6451628
references.citation: Nat Struct Biol. 1999 Oct;6(10):976-83
references.pmid: 10504734
references.citation: Biochemistry. 2006 Sep 19;45(37):11140-50
references.pmid: 16964975
references.citation: Proc Natl Acad Sci U S A. 2004 Sep 7;101(36):13105-10
references.pmid: 15340147
references.citation: Protein Sci. 2007 Oct;16(10):2287-93
references.pmid: 17893365
references.citation: Biochemistry. 1996 Sep 24;35(38):12241-50
references.pmid: 8823157
references.citation: Eur J Biochem. 2002 Apr;269(7):1905-15
references.pmid: 11952792
references.citation: Biophys J. 2005 Nov;89(5):3312-9
references.pmid: 16100265
references.citation: J Synchrotron Radiat. 1996 Jul 1;3(Pt 4):185-96
references.pmid: 16702677
references.citation: J Am Chem Soc. 2004 Apr 21;126(15):4991-5000
references.pmid: 15080705
references.citation: Chem Rev. 2004 Feb;104(2):1047-76
references.pmid: 14871149
references.citation: Cell Mol Life Sci. 2000 Aug;57(8-9):1236-59
references.pmid: 11028916
references.citation: J Biol Chem. 1991 Sep 15;266(26):16996-7003
references.pmid: 1894598
references.citation: Inorg Chem. 2011 Jan 17;50(2):482-8
references.pmid: 21141901
references.citation: J Biol Inorg Chem. 2009 Aug;14(6):905-12
references.pmid: 19381697
references.citation: J Biol Inorg Chem. 2010 Sep;15(7):1033-49
references.pmid: 20437064
references.citation: J Biol Chem. 2007 Dec 7;282(49):35695-702
references.pmid: 17893146
references.citation: J Am Chem Soc. 2007 Jan 24;129(3):709-18
references.pmid: 17227035
references.citation: J Mol Biol. 1986 Nov 20;192(2):361-87
references.pmid: 3560221
references.citation: Biochemistry. 1995 Mar 7;34(9):2857-65
references.pmid: 7893699
references.citation: Biochemistry. 1997 Sep 9;36(36):10901-9
references.pmid: 9283080
references.citation: Biochemistry. 2004 May 18;43(19):5735-47
references.pmid: 15134448
references.citation: Biochemistry. 1999 Nov 16;38(46):15086-96
references.pmid: 10563791
references.citation: J Biol Chem. 1991 Dec 5;266(34):23120-7
references.pmid: 1744110
references.citation: J Biol Chem. 2003 Dec 12;278(50):49691-8
references.pmid: 12966104
references.citation: J Biol Inorg Chem. 2000 Jun;5(3):341-53
references.pmid: 10907745
references.citation: J Biol Chem. 2006 Mar 31;281(13):8981-90
references.pmid: 16436386
references.citation: J Biol Chem. 2006 Feb 17;281(7):4190-8
references.pmid: 16330540