期刊
PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
卷 58, 期 1, 页码 14-21出版社
WILEY
DOI: 10.1002/prot.20293
关键词
tetrahydrobiopterin; protein stability; kinetics; human; Parkinsonism
资金
- NIGMS NIH HHS [GM47291, R01 GM047291] Funding Source: Medline
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM047291] Funding Source: NIH RePORTER
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 7.2degrees for the T245P, R306H, and T463M enzymes, while the T283M enzyme was too unstable for measurement of a T. 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. (C) 2004 Wiley-Liss, Inc.
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