苯丙氨酸脱氢酶的突变.pdfVIP

11390 Biochemistry 2002, 41, 1139011397 Single Amino Acid Substitution in Bacillus sphaericus Phenylalanine Dehydrogenase Dramatically Increases Its Discrimination between Phenylalanine and Tyrosine Substrates Stephen Y. K. Seah,‡,⊥ K. Linda Britton,§ David W. Rice,§ Yasuhisa Asano, and Paul C. Engel*,‡ Department of Biochemistry and Conway Institute of Biomolecular and Biomedical Research, Uni ersity College Dublin, Belfield, Dublin 4, Ireland, Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, Uniersity of Sheffield, P.O. Box 594, Sheffield S10 2UH, United Kingdom, and Biotechnology Research Center, Toyama Prefectural Uni ersity, 5180 Kurokawa, Kosugi, Toyama 939-03, Japan Recei ed March 12, 2002; Reised Manuscript Receied May 20, 2002 ABSTRACT : Homology-based modeling of phenylalanine dehydrogenases (PheDHs) from various sources, . ). s using the structures of homologous enzymes Clostridium symbiosum glutamate dehydrogenase and Bacillus e C l T c sphaericus leucine dehydrogenase as a guide, revealed that an asparagine residue at position 145 of B. i t U r ( a 0 d sphaericus PheDH was replaced by valine or alanine in PheDHs from other sources. This difference was 4 e : h proposed to be the basis for the poor discrimination by the B. sphaericus enzyme between the substrates 6 s 4 i : l 2 b L-phenylalanine and L-tyrosine. Residue 145 of this enzyme was altered, by site-specific mutagenesis, to 1 u t p