EXPRESSION IN YEAST OF 3 ALLELIC CDNAS CODING FOR HUMAN LIVER P-450 3A4 - DIFFERENT STABILITIES, BINDING-PROPERTIES AND CATALYTIC ACTIVITIES OF THE YEAST-PRODUCED ENZYMES

Three natural allelic cDNAs coding for P-450 3A4, the major form in hu man liver, namely NF25, NF10 and hPCN1, have been expressed in Sacchar omyces cerevisiae. NF25 and hPCN1 were functionally expressed in yeast microsomes, yielding proteins with an absorption maximum at 448 nm in the CO-reduced difference spectrum. Some catalytic activities and sub strate binding properties of P-450 NF25 and P-450 hPCN1 in yeast micro somes have been compared; no striking difference was found, showing th at the two point substitutions between their amino-acid sequences (Trp 392 and Thr431 in P-450 NF25 are replaced by Val392 and Ile431 in P-45 0 hPCN1) have no significant effect on the functional properties of th ese two variants. By contrast, P-450 NF10, which differs from P-450 NF 25 by a one-amino-acid deletion (Ile224 replacing Thr224-Val225), was produced as a denatured form, as revealed by an absorption maximum at 420 nm, and was not catalytically active. This suggests that the delet ion prevents the correct folding of the protein. The results of this s tudy show that P-450 NF25 and P-450 hPCN1 are two roughly equivalent, functionally active variants of P-450 3A4, but that P-450 NF10 is a de fective, unstable gene product that could arise from an alternative mR NA splicing. This could contribute to the large variations reponed for nifedipine oxidation, a typical P-450 3A4 activity, in human liver.