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Volume 271, Number 45, Issue of November 8, 1996 pp. 28607-28611
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Different Structural Requirements at Specific Proline Residue Positions in the Conserved Proline-rich Region of Cytochrome P450 2C2

(Received for publication, July 22, 1996, and in revised form, August 28, 1996)

From the Department of Molecular and Integrative Physiology, College of Medicine at Urbana-Champaign, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Cytochrome P450 is anchored to the endoplasmic reticulum membrane by an N-terminal transmembrane sequence with the catalytic domain facing the cytoplasmic side. Within the peptide sequence linking these two domains is a highly conserved proline-rich region. In cytochrome P450 2C2, this region has the sequence ³⁰PPGPTPFP³⁷. To examine the structural requirements at these proline residues, each proline was replaced with alanine, glycine, valine, or an acidic amino acid, and the activities of the mutated proteins were determined in transfected COS-1 cells. Lauric acid 1-hydroxylase activities of Pro³⁰ and Pro³³ mutants were less than 10% of wild type for each substitution except for alanine, which was 25-30%. In striking contrast, substitutions at Pro³¹, including an acidic residue, did not substantially alter activity. At positions 35 and 37, acidic amino acid substitutions reduced activity to less than 10% of wild type while substitution of the other three amino acids had little effect. The tolerance of substitutions of charged residues at Pro³¹ suggests that the side chain at this position is exposed to a polar environment; conversely, the reduced activity with charged substitutions, but not with uncharged substitutions at positions 35 and 37, suggests that these residues are exposed to a hydrophobic environment, presumably within the folded protein. The loss of activity with substitutions at Pro³⁰ and Pro³³ implies that the motif PXXP is important for the formation of a functional cytochrome P450 and that this sequence might have a helical structure with a repeat of three, as in the left-handed poly-L-proline II helix. Insertion of alanine between positions 29 and 30 did not substantially affect activity, but insertions between either 33 and 34 or 37 and 38 resulted in activity less than 25% of wild type. These data indicate that the position of PXXP, relative to the sequence flanking it on the C-terminal side, may be important for its function.

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