Mutations of P450c21 (steroid 21-hydroxylase) at Cys⁴²⁸, Val²⁸¹, and Ser²⁶⁸ result in complete, partial, or no loss of enzymatic activity, respectively

Steroid 21-hydroxylase deficiency is the major cause of congenital adrenal hyperplasia (CAH), a common genetic disease. To define the relationship between gene mutations and enzyme deficiency, we generated missense mutations of the 21-hydroxylase cDNA at three different sites and characterized the mutant proteins after expressing them in cultured mammalian and yeast cells. Among them, Ser²⁶⁸ and Val²⁸¹ have been found to be mutated in CAH patients, whereas Cys⁴²⁸ has been implicated as the heme ligand. Our results show mutations at these sites result in complete, partial, or no loss of the enzymatic activity. All the Cys⁴²⁸ mutants had neither enzymatic activity nor P450 absorption, thus supporting the notion that Cys⁴²⁸ is the heme ligand. All the 268-mutants exhibited the same activity as normal 21-hydroxylase, demonstrating that the clinically observed Ser²⁶⁸ → Thr change represents a polymorphism rather than the cause of the enzyme deficiency. The 281-mutants had normal K_m but greatly reduced V_max values that also paralleled the reduction in the heme content, in the order Val²⁸¹ (normal, 100%) > Ile²⁸¹ (50%) > Leu²⁸¹ (20%) > Thr²⁸¹ (10%). Our findings suggest that the methyl group at the β-carbon of Val²⁸¹ is required for heme incorporation and consequently enzymatic activity.