REQUIREMENT OF PHENYLALANINE-343 FOR THE PREFERENTIAL DELTA-4-LYASE VERSUS DELTA-5-LYASE ACTIVITY OF RAT CYP17

Site-directed mutagenesis of a domain (amino acids 343-348) within the conserved region of rat CYP17 was performed to investigate species-sp ecific differences between rat and human/bovine DELTA4-versus DELTA5-l yase activity. This domain displays substantial deviations between the rat and human/bovine/pig sequences and includes Arg346, which is know n to he essential for DELTA4-lyase (Kitamura, M., Buczko, E., and Dufa u, M. L. (1991) Mol. Endocrinol. 5, 1373-1380) and DELTA5-lyase activi ties. Analysis of the DELTA4-lyase activity of mutant rat CYP17 cDNA e xpressed in nonsteroidogenic COS-1 cells revealed that substitution of Phe at position 343 in the rat with Ile of the human/bovine sequence resulted in a reduction in DELTA4-lyase activity to levels in the rang e of the DELTA5-supported reaction. This Phe343 --> Ile mutant Cyp17 d id not exhibit changes either in DELTA5-supported lyase activity or in DELTA4- and DELTA5-hydroxylase activities. Substitution of Asn344, Se r347, and His348 in rat CYP17 with the corresponding bovine amino acid s Ser, Asn, and Arg did not enhance this effect. Thus, the reduced act ivity of the bovine CYP17 DELTA4-lyase reaction can be mimicked in par t in the rat polypeptide by the substitution of Phe343 with the bovine counterpart, Ile. Unlike the bovine CYP17-catalyzed reaction, the rat Phe343 --> Ile mutant exhibited a low level lyase activity (k(cat)) t hat did not discriminate between DELTA4- and DELTA5-substrates. These results suggest that the presence of Phe343 enhances the DELTA4-suppor ted lyase activity possibly through stabilization of a DELTA4-specific interaction.