A CONSERVED PROLINE-RICH SEQUENCE BETWEEN THE N-TERMINAL SIGNAL-ANCHOR AND CATALYTIC DOMAINS IS REQUIRED FOR ASSEMBLY OF FUNCTIONAL CYTOCHROME-P450 2C2

In cytochrome P450 2C2, the region which links the N-terminal signal a nchor with the catalytic domain contains a highly conserved proline-ri ch region with the sequence, 30-PPGPTPFP-37. Mutation of proline-30 or proline-33 diminished activities of the mutants expressed in COS-1 ce lls (Chen, C., and Kemper, B. (1996) J. Biol. Chem. 271, 28697-28611). Substitution of alanine, proline, or arginine for glycine-32 abolishe d laurate hydroxylase activity of the proteins expressed in COS-1 cell s, which suggests that this residue is also functionally important. To determine the basis for the decreased activity in COS-1 cells, the ac tivities and spectral properties of mutant proteins expressed in insec t cells and bacteria were determined. Substitution of alanine for eith er proline-30 or -33 resulted in reduced expression in insect cells of functional cytochrome P450 hemoprotein and an increase in the express ion of inactive cytochrome P420. In contrast, substitution of alanine for proline-31, -35, or -37 resulted in hemoproteins with spectra simi lar to cytochrome P450 2C2 so that the amount of cytochrome P450 expre ssed in insect cells correlated with the activities of the mutants in COS-1 cells. The laurate hydroxylase activities per nanomole of cytoch rome P450 in insect microsomes were similar for wild type and all muta nts, indicating that, once folded, the catalytic activity of membrane- bound cytochrome P450 was not affected by the mutations. Expression in bacteria resulted in diminished expression of cytochrome P450 for all mutants, with the greatest decrease for the proline-30 and -33 mutant s, and increased cytochrome P420. In contrast to the insect cell studi es, the proline-30 and -33 mutants were inactive, while the other muta nts had specific activities 30-70% of cytochrome P450 2C2. These data are consistent with a role for the proline-rich region in efficient as sembly of cytochrome P450 2C2 in eukaryotic cells. Mutations of this r egion also may affect the conformational integrity of the proteins, wh ich was revealed by assays of solubilized bacterially expressed protei ns. (C) 1998 Academic Press.