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The role of tyrosines 223 and 238 in Rhodotorula gracilisd-amino acid oxidase catalysis: Interpretation of double mutations

Paper ID Volume ID Publish Year Pages File Format Full-Text
18397 42721 2006 8 PDF Available
Title
The role of tyrosines 223 and 238 in Rhodotorula gracilisd-amino acid oxidase catalysis: Interpretation of double mutations
Abstract

The active site of flavoprotein oxidases frequently contains two active-site tyrosines, e.g., Y223 and Y238 in d-amino acid oxidases (DAAO) from Rhodotorula gracilis. In the past, these residues were individually mutated to phenylalanine and serine. To further study their role we undertake an interpretation of Y223F–Y238F double mutation. The spectral properties of the double mutant are similar to those of wild-type DAAO, suggesting a minimally altered active site, but its catalytic activity is lower. A first unexpected observation is that for a number of properties the double mutant is more similar to the wild-type DAAO than to the corresponding single-point mutants, e.g., similar kinetic mechanism, substrate specificity, and redox properties. The Y223F–Y238F DAAO also exhibits appreciably weaker binding of the competitive inhibitors’ carboxylic acids (synergistic effects), while the Km values for the substrate d-amino acids are only slightly changed, thus suggesting that the presence of an amino group in the ligand is important for binding to the double mutant. Synergistic effects of the substitutions are also evident on the rate constant of flavin reduction indicating anti-cooperative interaction of the two residues in the hydride transfer process. Our results indicate that Tyr223-OH and Tyr238-OH contribute to the high catalytic efficiency of DAAO allowing the precise alignment required for optimal catalysis.

Keywords
E-FADox, oxidized enzyme; E-FADred, reduced enzyme; E-FADseq, semiquinone enzyme; IA, imino acid product; Enzymes, D-amino acid oxidase (DAAO, EC 1.4.3.3); pkDAAO, pig kidney D-amino acid oxidase; RgDAAO, Rhodotorula gracilis D-amino acid oxidaseFlavoprot
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The role of tyrosines 223 and 238 in Rhodotorula gracilisd-amino acid oxidase catalysis: Interpretation of double mutations
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 38, Issue 6, 1 April 2006, Pages 795–802
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us