MOLECULAR BIOLOGY OF THE PTERIN-DEPENDENT HYDROXYLASES AND ANCILLARY ENZYMES

我们对芳香族氨基酸羟基酶和所需酶的攻击 为了合成和维持辅因子四氢生物蝶呤，已经 是多层次的。我们已经克隆了几个酶的cDNA 系统。这些克隆包括一组用来扫描DNA或RNA的探针 从精神疾病患者身上提取样本以识别未知突变 可能与这些疾病的某些子集有关的基因。 此外，我们对这些酶的克隆和表达已经产生了 适合生化研究的蛋白质数量。我们正在申请所有 已知这些酶的催化和调节位点以及 表达了几种带有定向改变的酶。在这 方式，我们一直在测试我们关于重要领域的想法 确定这些位点在催化、亲和力方面发挥的作用 底物和效应器分子等。 例如，我们已经表达了我们的大鼠sepiapterin的cdna克隆。 还原酶，一种合成辅因子所必需的酶 四氢生物蝶呤(芳香族氨基羟化所必需的 酸)，在细菌中获得了大量的活性酶。这 中任何酶的第一个克隆(来自任何生物) 四氢生物蝶呤的生物合成途径，这是必需的 多巴胺和5-羟色胺能神经递质的生物合成。

Our attack on the aromatic amino acid hydroxylases and the enzymes required for synthesis and maintenance of the cofactor, tetrahydrobiopterin, has been multi-level. We have cloned several of the cDNAs for enzymes in this system. These clones comprise a battery of probes to scan DNA or RNA samples from individuals with mental diseases to identify unknown mutations in genes that may be responsible for some subset of these disorders. Additionally, our cloning and expression of these enzymes has produced quantities of protein suitable for biochemical study. We are applying all that is known about catalytic and regulatory sites to these enzymes and have expressed several enzymes carrying directed alterations. In this manner, we have been testing our ideas about significant domains to determine the roles that these sites play in catalysis, affinity to substrates and effector molecules, etc. For example, we have expressed our cDNA clone for rat sepiapterin reductase, an enzyme essential for synthesis of the cofactor tetrahydrobiopterin (essential for the hydroxylation of aromatic amino acids), in bacteria and obtained a large quantity of active enzyme. This represents the first clone (from any organism) for any enzyme in the tetrahydrobiopterin biosynthetic pathway, which is necessary for the biosynthesis of dopaminergic and serotinergic neurotransmitters.