title: Expression and characterization of human bifunctional peptidylglycine alpha-amidating monooxygenase.
abstract: We report the purification and characterization of human bifunctional peptidylglycine alpha-amidating monooxygenase (the bifunctional PAM) expressed in Chinese hamster ovary cells. PAM is in charge of the formation of the C-terminal amides of biologically active peptides. The bifunctional PAM possesses two catalytic domains in a single polypeptide, peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PAL, EC 4.3.2.5). By introducing a stop codon at 835 Glu, we were able to eliminate the membrane-spanning domain in the C-terminal region and succeeded in purifying a soluble form of bifunctional PAM that was secreted into the medium. Through a three-step purification procedure, we obtained 0.3mg of the purified PAM, which showed a single band at 91 kDa on SDS-PAGE, from 1L of monolayer culture medium. Metals contained in the purified PAM were analyzed and chemical modifications were performed to gain insight into the mechanism of the PAL reaction. Inductively coupled plasma detected 0.62 mol of Zn(2+) and 1.25 mol of Cu(2+) per mol of bifunctional PAM. Further, the addition of 1mM EDTA reduced the PAL activity by about 50%, but the decreased activity was recovered by the addition of an excess amount of Zn(2+). In a series of chemical modifications, phenylglyoxal almost completely eliminated the PAL activity and diethyl pyrocarbonate suppressed activity by more than 70%. These findings implied that Arg and His residues might play crucial roles during catalysis.
authors.affiliation: Department of Medical Biochemistry, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Satani
authors.forename: Kenichi
authors.initials: K
authors.lastname: Takahashi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sakamoto
authors.forename: Saori
authors.initials: S
authors.lastname: Harada
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Kaida
authors.forename: Masato
authors.initials: M
authors.lastname: Noguchi
mesh_terms: D019760:Amidine-Lyases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002384:Catalysis / Q000187:drug effects; D003300:Copper / Q000494:pharmacology; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology
chemical_list: D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D003300:Copper; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D019760:Amidine-Lyases; C068521:peptidylamidoglycolate lyase; D015032:Zinc