Full structure of Human 21-Hydroxylase, showing three identical subunits, each with a centralized heme group (magenta)
21-hydroxylase is a complex of three independent and identical enzyme subunits. Each subunit in the human enzyme consists of 13 α-helices and 9 ß-strands, formed into a triangular prism-like tertiary structure. The iron(III) heme group that defines the active site resides in the center of each subunit. The human enzyme binds one substrate at a time. In contrast, the well-characterized bovine enzyme can bind two substrates. The human and bovine enzyme share 80% amino acid sequence identity, but are structurally different, particularly in loop regions, and also evident in secondary structure elements.
21-Hydroxylase is highly specific for hydroxylation of progesterone and 17-hydroxyprogesterone. No studies have reported sufficient binding of alternate substrates. In this way, it differs from the evolutionarily and functionally related P450 enzyme 17-hydroxylase, which has a large range of substrates.
Earlier studies of the human enzyme expressed in yeast classified 17-hydroxyprogesterone as the best substrate for 21-hydroxylase. However, recent analysis of the purified human enzyme found a lower KM and greater catalytic efficiency for progesterone over 17-hydroxyprogesterone.
The 2015 analysis found the catalytic efficiency of 21-hydroxylase for conversion of progesterone in humans to be approximately 1.3 x 10^7 M-1s-1 at 37 °C. This makes it the most catalytically efficient P450 enzyme of those reported, as of 2015, and more catalytically efficient than the closely related bovine 21-hydroxylase enzyme. C-H bond breaking to create a primary carbon radical is thought to be the rate-limiting step in the hydroxylation.
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Human steroidogenesis, showing both reactions of 21-Hydroxylase at center top.
Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder, and occurs in approximately 1 in 15000 births globally. There are multiple forms of CAH, broken down into classical and nonclassical forms based on the amount of function retained. The classical forms include salt-wasting (SW), and simple-viralizing (SV). Mutations that interfere with the active site—the heme group or residues involved in substrate binding—result in a complete loss of enzymatic activity, the salt-wasting type. Cortisol and aldosterone deficits are associated with life-threatening salt-loss (hence salt-wasting), as the steroids play roles in regulating sodiumhomeostasis. Retaining minimal enzyme activity, the simple-viralizing type is associated with mutations in conserved hydrophobic regions or near the transmembrane domain. Simple viralizing CAH patients maintain adequate sodium homeostasis, but exhibit other phenotypical symptoms shared by SW, including accelerated growth in childhood and ambiguous genitalia in female neonates. Nonclassical forms retain 20-60% of hydroxylase function—this form is associated with normal cortisol expression, but an excess of androgens post-puberty.
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