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Biochim Biophys Acta. 1997 Apr 4;1338(2):259-66.

Functional interaction between amino-acid residues 242 and 290 in cytochromes P-450 2B1 and 2B11.

Author information

1
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721, USA. harlow@tonic.pharm.arizona.edu

Abstract

Previous studies have revealed the functional importance of the negatively charged amino-acid residue Asp-290 of the phenobarbital-inducible dog liver cytochrome P-450 (P-450) 2B11 (Harlow, G.R. and Halpert J.R. (1996) Arch. Biochem. Biophys. 326, 85-92). A search for P-450 2B11 residues capable of forming a charge pair with Asp-290 suggested the positively charged residue Lys-242 as a likely candidate. Replacement of Lys-242 with Asp in a P-450 2B11 fusion protein with rat NADPH-cytochrome P-450 reductase (reductase) resulted in very low holoenzyme expression levels in Escherichia coli, as did replacement of Asp-290 with Lys. Remarkably, however, expression levels of the double mutant Lys-242 --> Asp/Asp-290 --> Lys were dramatically increased above either single replacement alone. Similarly, the pair-wise substitutions Lys-242 --> Leu/Asp-290 --> Ile in P-450 2B11 and Leu-242 --> Lys/Ile-290 --> Asp in P-450 2B1 showed greater holoenzyme expression levels than the constituent single mutants, providing further evidence for the close proximity of these residues within the three-dimensional structure of these two enzymes. These results support the hypothesis that a functional interaction exists between residues 242 and 290, which may help to coordinate the relative positions of proposed helices G and I. All of the mutant combinations, including the additional P-450 2B11 double mutants Tyr-242/Asn-290 and Tyr-242/Ser-290, displayed altered stereoselectivity of androstenedione hydroxylation.

PMID:
9128144
DOI:
10.1016/s0167-4838(96)00209-9
[Indexed for MEDLINE]

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