CRBP mutants: relationship between retinol binding and efficiency as substrate for retinal synthesis

P. Penzes*, J. L. Naooli

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Two enzymes generate alWrans-retinoic acid (RA) from retinol, retinol dehydrogenase (RoDH) and retinal dehydrogenase. RoDH recognizes holocellular rettnol-binding protein (CRBP), which binds retinol tightly in a solventinaccessible pocket. Residues from the "portal" region of CRBP may be involved in this interaction and in mediating structural changes in CRBP that allow access of RoDH to retinol. To test these suppositions point mutations were introduced into CRBP: L29A, 132E, L35A, L35E, L35R, L36A, F57A, R58A and R58E. These mutants bind retinol with affinities 2 to 5-fold lower than the wild type, except Leu35 mutants which have similar IQ values as the wild type. All mutants show a conformation of retinol in the binding pocket similar to wild type, as reflected by UV spectra. Sensitivities to digestion by Arg-C were inversely proportional to affinities for retinol. V""/K" values of RoDH with mutants ranged from 0.7 to 2-fold of the wild type, and a linear relationship was observed between V-4/Kn, and retinol binding affinities, with some notable exceptions. In the exceptions, L29A, L36A, and R58E, lower binding affinity did not enhance the Vnuu/Kn, as much as in other mutants, possibly because the changes interfere with the ability of RoDH to access retinol. These data show that the efficiency of CRBP as substrate for RoDH does not depend solely on its affinity for retinol, and that amino acid residues contribute differentially to retinol binding and enzyme recognition. The portal residues contribute significantly to overall ligand binding, reflected by a cumulative 2500-fold decrease in 1Q caused by their.

Original languageEnglish (US)
Pages (from-to)A884
JournalFASEB Journal
Issue number9
StatePublished - Dec 1 1997

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics


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