Journal
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Volume 424, Issue 1, Pages 33-43Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.abb.2003.12.040
Keywords
cytochrome P450; site-directed mutagenesis; structure-function relationships; substrate specificity; alkoxyresorufin 0-dealkylation; molecular modeling; molecular dynamics
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Funding
- NCI NIH HHS [CA85542] Funding Source: Medline
- NCRR NIH HHS [RR16440] Funding Source: Medline
- NIEHS NIH HHS [ES07628] Funding Source: Medline
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Five reciprocal active site mutants of P450 1A1 and 1A2 and an additional mutant, Val/Leu-382 --> Ala, were constructed, expressed in Escherichia coli, and purified by Ni-NTA affinity chromatography. In nearly every case, the residue replacement led to loss of 7-methoxy- and 7-ethoxyresorufin O-dealkylase activity compared to the wild-type enzymes, except for the P450 1A1 S122T mutation which increased both activities. Mutations at position 382 in both P450 1A1 and 1A2 shifted substrate specificity from one enzyme to another, confirming the importance of this residue. Changes in activity of P450 1A enzymes upon amino acid replacement were, in general, consistent with molecular dynamics analyses of substrate motion in the active site of homology models. (C) 2004 Elsevier Inc. All rights reserved.
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