IDENTIFICATION OF TOPAQUINONE AND ITS CONSENSUS SEQUENCE IN COPPER AMINE OXIDASES

被引:190
作者
JANES, SM
PALCIC, MM
SCAMAN, CH
SMITH, AJ
BROWN, DE
DOOLEY, DM
MURE, M
KLINMAN, JP
机构
[1] UNIV CALIF BERKELEY,DEPT CHEM,BERKELEY,CA 94720
[2] UNIV ALBERTA,DEPT FOOD SCI,EDMONTON T6G 2P5,ALBERTA,CANADA
[3] STANFORD UNIV,BECKMAN CTR,STANFORD,CA 94305
[4] AMHERST COLL,DEPT CHEM,AMHERST,MA 01002
关键词
D O I
10.1021/bi00163a025
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The nature of the active site cofactor and the amino acid sequence flanking this structure have been determined in a range of copper amine oxidases. For enzymes from porcine plasma, porcine kidney, and pea seedlings, proteolytic digestion was performed on phenylhydrazone or p-nitrophenylhydrazone derivatives. Thermolysin treatment leads to relatively small active site peptides, which have been characterized by Edman degradation and by resonance Raman spectroscopy. Resonance Raman spectra of peptides show identical peak positions and intensities relative to each other and to a model p-nitrophenylhydrazone derivative of topaquinone hydantoin, establishing topaquinone as the cofactor in each instance. Edman degradation of peptides provides active site sequences for comparison to previous determinations with bovine serum and yeast amine oxidases. The available data establish a consensus sequence of Asn, Topa, Asp/Glu. Trypsin leads to significantly longer peptides, which reveal a high degree of sequence identity between plasma proteins from bovine and porcine sources (89%), with significantly decreased identity between the porcine serum and intracellular amine oxidases (56%). A lower degree of identity (45%) is observed between the pea seedling and mammalian enzymes. As an alternative to the isolation of active site peptides for topaquinone identification, visible spectra of intact proteins have been investigated. It is shown that p-nitrophenylhydrazone derivatives of native enzymes, active site-derived peptides, and a topaquinone model exhibit identical behavior, absorbing at 457-463 nm at neutral pH (pH 7.2) and at 575-587 nm in basic solution (1-2 M KOH). These spectral properties, which appear unique to topaquinone, provide a rapid and simple test for the presence of this cofactor in intact enzymes. Using this approach, the class of enzymes demonstrated to contain topaquinone has been expanded to include copper amine oxidases from sheep plasma and chick pea seedling.
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收藏
页码:12147 / 12154
页数:8
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