Identification of the quinone cofactor in mammalian semicarbazide-sensitive amine oxidase.
Mammalian semicarbazide-sensitive amine oxidase (SSAO) enzymes have been classified as EC 1.4.3.6 [amine:oxygen oxidoreductase (deaminating)(copper-containing)]. However, both the identity of the quinone cofactor and the presence of copper remain unconfirmed, and SSAO has proved impossible to purify to homogeneity in sufficient yield to permit cofactor identification. To circumvent this problem, we have partially purified SSAO enzymes from bovine and porcine aortae and have established, with a redox-cycling assay, that no other quinoproteins were present in enzyme preparations. Enzymes were then derivatized with (p-nitrophenyl)hydrazine (p-NPH), which forms a covalent yellow complex with the quinone cofactor. Visible absorbance spectra of derivatized bovine and porcine enzymes (respective lambdamax values 456 and 476 nm at neutral pH, shifting to 580 and 584 nm in 2 M KOH) were consistent with the presence of (2,4,5-trihydroxyphenyl)alanine quinone (TPQ) as cofactor. Resonance Raman spectra were essentially identical to that for pea seedling amine oxidase, a known TPQ-containing enzyme. Extensive digestion of SSAO enzymes, and of porcine kidney diamine oxidase, with pronase E yielded species with identical chromophoric properties characteristic of the dipeptide, TPQ(p-NPH)-Asp. Thermolytic digestion of porcine SSAO gave two cofactor-containing peptides that contained a TPQ consensus sequence, Asn-X-Asp-Tyr-Tyr, where X is a blank cycle corresponding to TPQ. N-terminal sequencing of whole enzymes revealed a membrane-spanning region typical of an extracellular type II glycoprotein. These results confirm the presence of TPQ in mammalian membrane-bound SSAO ectoenzymes.
Holt
A
A
Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
Alton
G
G
Scaman
C H
CH
Loppnow
G R
GR
Szpacenko
A
A
Svendsen
I
I
Palcic
M M
MM
D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000201:enzymology; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003067:Coenzymes / Q000737:chemistry* / Q000378:metabolism; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D013059:Spectrum Analysis, Raman; D013552:Swine
C086758:2,4,5-trihydroxyphenylalanine quinone; D003067:Coenzymes; D010446:Peptide Fragments; D004295:Dihydroxyphenylalanine; D006631:Amine Oxidase (Copper-Containing)