Effects of mutations in tyrosine hydroxylase associated with progressive dystonia on the activity and stability of the protein

Abstract

Tyrosine hydroxylase (TyrH) catalyzes the conversion of tyrosine to dihydroxyphenylalanine (DOPA), the rate-limiting step in the biosynthesis of dopamine. Four mutations in the TyrH gene have recently been described in cases of autosomal recessive DOPA-responsive dystonia (Swaans et al., Ann Hum Genet 2000;64:25-31). All four are predicted to result in changes in single amino acid residues in the catalytic domain of the protein: T245P, T283M, R306H, and T463M. To determine the effects of these mutations on the molecular properties of the enzyme, mutant proteins containing the individual single amino acid changes have been expressed in bacteria and purified. Only the T283M mutation results in a decrease in the enzyme k(cat) value, while the T245P enzyme has a slightly higher value than the wild-type enzyme. The only case in which a K(m) value for either tyrosine or tetrahydrobiopterin is perturbed is the T245P enzyme, for which the K(m) value for tyrosine has increased about 50%. In contrast to the minor effects of the mutations on enzyme activity, the stability is decreased significantly by the mutations. The R306H and T283M enzymes are the least stable, losing activity 30- and 50-fold more rapidly than the wild-type enzyme. The apparent T(m) value for unfolding was decreased by 3.9, 8.2, and 7.2 degrees for the T245P, R306H, and T463M enzymes, while the T283M enzyme was too unstable for measurement of a T(m) value. The results establish that the physiological effects of the mutations are primarily due to the decreased stability of the mutant proteins rather than decreases in their intrinsic activities.

Discovery of compounds that protect tyrosine hydroxylase activity through different mechanisms

“…THD-associated mutations correlate with loss-of-function-misfolding and instability of TH [14][15][16], and the mutants are rapidly degraded by intracellular proteases, leading to decreased TH and dopamine levels in THD patients. Increasing evidence points to pharmacological chaperones as a promising therapeutic strategy for loss-offunction misfolding diseases [17][18][19][20].…”

Canine Tyrosine Hydroxylase (TH) Gene and Dopamine .BETA.-Hydroxylase (DBH) Gene: Their Sequences, Genetic Polymorphisms, and Diversities among Five Different Dog Breeds

“…The effect of silent SNPs should also be analyzed [4]. Since the locations of all SNPs identified in this study were different from those of SNPs in human TH and DBH genes, possible structural and functional changes derived from these SNPs should be examined as have been reported for human genes [7,33,34]. With regards to the breed differences of these SNPs, C97T on the TH gene and A1819G on the DBH gene are only found in Shibas.…”

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“…Thus far, several deletion and missense mutations, more than 20 noncoding SNPs, and various synonymous coding and nonsynonymous coding SNPs, a splice junction mutation, and at least three promoter mutations have been reported in the TH gene (Supplementary Table S1). Expression analysis and biochemical characterization of the missense mutant enzymes have revealed that they have either normal or decreased enzymatic activity (V max ) in vitro, but all seem to have decreased thermal stability in vitro [Knappskog et al, 1995;Ludecke et al, 1996;Royo et al, 2005], similar to what has been found for the ''mild'' variants of PAH associated with PKU (www.pahdb.mcgill.ca).…”