The hyt/hyt hypothyroid mouse has an autosomal recessive, fetal-onset, severe hypothyroidism related to TSH hyporesponsiveness and associated with elevated TSH. Our previous work has suggested that the hypothyroidism and TSH hyporesponsiveness may result from a mutation in the hyt/hyt TSH receptor (TSHr) of the thyroid gland. Based on DNA sequencing of the entire coding region of the TSHr gene from the wild-type BALB/cBY +/+ mouse, the +/+ TSHr is 92% and 94% identical at the nucleotide and amino acid residue levels, respectively, compared to the rat TSHr gene. The coding region of the hyt/hyt TSHr, compared to that of the +/+ TSHr, has a single base change, CCG to CTG, at nucleotide position 1666, which leads to the replacement of a highly conserved proline at amino acid position 556 with a leucine in transmembrane domain IV. This mutation was introduced by site-directed mutagenesis into the wild-type human TSHr and transiently expressed in COS-7 cells. Although the size and abundance of the mutant TSHr mRNA suggested that there was no effect on the nature of the mRNA, TSH binding and the response to TSH in transfected cells were abolished. Further studies are necessary to clarify how the Pro to Leu replacement interferes with receptor expression on the cell surface or influences TSH binding. These functional consequences of the mutation appear to account for the observed TSH hyporesponsiveness and hypothyroidism in the hyt/hyt mouse.

Pulmonary cytochrome P450 2F3 (CYP2F3) catalyzes the dehydrogenation of the pneumotoxin 3-methylindole (3MI) to an electrophilic intermediate, 3-methyleneindolenine, which is responsible for the toxicity of the parent compound. Members of the CYP2F subfamily are the only enzymes known to exclusively dehydrogenate 3MI, without detectable formation of oxygenation products. Thus, CYP2F3 is an attractive model to study dehydrogenation mechanisms. The purpose of this study was to identify specific residues that could facilitate 3MI dehydrogenation. Both single and double mutations were constructed to study the molecular mechanisms that direct dehydrogenation. Double mutations in SRS 1 produced an inactive enzyme, while double mutants in SRS 4 did not alter 3MI metabolism. However, double mutations in SRS 5 and SRS 6 successfully introduced oxygenase activity to CYP2F3. Single mutations in SRS 5, SRS 6 and near SRS 2 also introduced 3MI oxygenase activity. Mutants S474H and D361T oxygenated 3MI but also increased dehydrogenation rates, while G214L, E215Q and S475I catalyzed 3MI oxygenation exclusively. A homology model of CYP2F3 was precisely consistent with specific dehydrogenation of 3MI via initial hydrogen atom abstraction from the methyl group. In addition, intramolecular kinetic deuterium isotope studies demonstrated an isotope effect (KH/KD) of 6.8. This relatively high intramolecular deuterium isotope effect confirmed the initial hydrogen abstraction step; a mutant (D361T) that retained the dehydrogenation reaction exhibited the same deuterium isotope effect. The results showed that a single alteration, such as a serine to isoleucine change at residue 475, dramatically switched catalytic preference from dehydrogenation to oxygenation.