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Reciprocal Size−Effect Relationship of the Key Residues in Determining Regio- and Stereospecificities of DHEA Hydroxylase Activity in P450 2a5
Reciprocal Size−Effect Relationship of the Key Residues in Determining Regio- and Stereospecificities of DHEA Hydroxylase Activity in P450 2a5
- Source :
- Biochemistry. 36:3193-3198
- Publication Year :
- 1997
- Publisher :
- American Chemical Society (ACS), 1997.
-
Abstract
- Collectively, the P450 2a4/2a5 system hyrdoxylates DHEA in at least three positions (7alpha, 7beta, and 2alpha). An individual P450, however, exhibits high specificity to one of these products. Using site-directed mutagenesis of mP450 2a5 from the wild mouse Mus minutoides and bacterial expression, we have associated the function of residues 117, 209, and 481 with the respective specificity observed in each P450. Ala at position 117 determines the 7beta-hydroxylase activity, whereas Val at this position defines the 2alpha-hydroxylase activity. Leu at position 209 is essential for high DHEA 7alpha-hydroxylase activity. The substitutions of residue 481 with various hydrophobic amino acids elicited a profound alteration of the specific hydroxylation rates, but did not influence the regio- and stereospecificities at either of the three positions of DHEA. The alterations caused by residue 481 also depended on the residue identity at position 117 or 209. The results indicate that the sizes of several key residues obey a concerted reciprocal relationship whereby the substrate pocket of the P450s adjusts to accommodate DHEA. A limited molecular modeling study successfully correlates DHEA binding to experimental DHEA hydroxylase activities for a series of mutants at key positions.
- Subjects :
- Stereochemistry
Molecular Sequence Data
Mutant
Stereoisomerism
Biochemistry
Mixed Function Oxygenases
Substrate Specificity
Cytochrome P-450 CYP2A6
Hydroxylation
Residue (chemistry)
chemistry.chemical_compound
Cytochrome P-450 Enzyme System
Escherichia coli
Animals
Amino Acid Sequence
Cloning, Molecular
Binding site
Peptide sequence
chemistry.chemical_classification
Binding Sites
Chemistry
Mutagenesis
Recombinant Proteins
Amino acid
Muridae
Kinetics
Liver
Oligodeoxyribonucleotides
Mutagenesis, Site-Directed
Aryl Hydrocarbon Hydroxylases
Subjects
Details
- ISSN :
- 15204995 and 00062960
- Volume :
- 36
- Database :
- OpenAIRE
- Journal :
- Biochemistry
- Accession number :
- edsair.doi.dedup.....d45a2b99b723227267dbaec435812b99
- Full Text :
- https://doi.org/10.1021/bi962654j