Bailey S W, Dillard S B, Thomas K B, Ayling J E
Department of Pharmacology, College of Medicine, University of South Alabama, Mobile 36688.
Biochemistry. 1989 Jan 24;28(2):494-504. doi: 10.1021/bi00428a013.
The structure of the cofactor binding domain of tyrosine hydroxylase (TH) was examined at physiological pH by determining kinetic parameters of (R)-tetrahydrobiopterin [(R)-BH4] and a series of tetrahydropterin (PH4) derivatives (6-R1-6-R2-PH4: R1 = H and R2 = methyl, hydroxymethyl, ethyl, methoxymethyl, phenyl, and cyclohexyl; R1 = methyl and R2 = methyl, ethyl, propyl, phenyl, and benzyl). A minimally purified TH preparation that was not specifically phosphorylated (designated as "unphosphorylated") was compared with enzyme phosphorylated with cAMP-dependent protein kinase. The Km for tyrosine with most tetrahydropterin analogues ranged between 20 and 60 microM with little decrease upon phosphorylation. Two exceptions were an unusually low Km of 7 microM with 6-ethyl-PH4 and a high Km of 120 microM with 6-phenyl-6-methyl-PH4, both with phosphorylated TH. Tyrosine substrate inhibition was elicited only with (R)-BH4 and 6-hydroxymethyl-PH4. With unphosphorylated TH (with the exception of 6-benzyl-6-methyl-PH4, Km = 4 mM) an inverse correlation between cofactor Km and side-chain hydrophobicity was observed ranging from a high with (R)-BH4 (5 mM) to a low with 6-cyclohexyl-PH4 (0.3 mM). An 8-fold span of Vmax was seen overall. Phosphorylation caused a 0.6-4-fold increase in Vmax and a 35-2000-fold decrease in Km for cofactor, ranging from a high of 60 microM with 6-methyl-PH4 to a low of 0.6 microM with 6-cyclohexyl-PH4. A correlation of the size of the hydrocarbon component of the side chain with affinity is strongly evident with phosphorylated TH, but in contrast to unphosphorylated enzyme, the hydroxyl groups in hydroxymethyl-PH4 (20 microM) and (R)-BH4 (3 microM) decrease Km in comparison to that of 6-methyl-PH4. Although 6,6-disubstituted analogues were found with affinities near that of (R)-BH4 (e.g., 6-propyl-6-methyl-PH4, 4 microM), they were frequently more loosely associated with phosphorylated TH than their monosubstituted counterparts (6-phenyl-PH4, 0.8 microM; cf. 6-phenyl-6-methyl-PH4, 8 microM). A model of the cofactor side-chain binding domain is proposed in which a limited region of nonpolar protein residue(s) capable of van der Waals contact with the hydrocarbon backbone of the (R)-BH4 dihydroxypropyl group is opposite to a recognition site for hydroxyl(s). Although interaction with either the hydrophilic or hydrophobic regions of unphosphorylated tyrosine hydroxylase is possible, phosphorylation by cAMP-dependent protein kinase appears to optimize the simultaneous operation of both forces.
通过测定（R）-四氢生物蝶呤[（R）-BH4]和一系列四氢蝶呤（PH4）衍生物（6-R1-6-R2-PH4：R1 = H且R2 = 甲基、羟甲基、乙基、甲氧基甲基、苯基和环己基；R1 = 甲基且R2 = 甲基、乙基、丙基、苯基和苄基）的动力学参数，在生理pH条件下研究了酪氨酸羟化酶（TH）辅因子结合结构域的结构。将未特异性磷酸化的最低限度纯化的TH制剂（称为“未磷酸化”）与用cAMP依赖性蛋白激酶磷酸化的酶进行比较。大多数四氢蝶呤类似物的酪氨酸Km值在20至60μM之间，磷酸化后降低很少。两个例外情况是，磷酸化的TH与6-乙基-PH4的Km异常低，为7μM，与6-苯基-6-甲基-PH4的Km高，为120μM。仅用（R）-BH4和6-羟甲基-PH4会引发酪氨酸底物抑制。对于未磷酸化的TH（6-苄基-6-甲基-PH4除外，Km = 4 mM），观察到辅因子Km与侧链疏水性之间呈负相关，范围从（R）-BH4的高值（5 mM）到6-环己基-PH4的低值（0.3 mM）。总体上观察到Vmax有8倍的跨度。磷酸化导致Vmax增加0.6至4倍，辅因子的Km降低35至2000倍，范围从6-甲基-PH4的高值60μM到6-环己基-PH4的低值0.6μM。对于磷酸化的TH，侧链烃成分的大小与亲和力之间的相关性非常明显，但与未磷酸化的酶相反，羟甲基-PH4（20μM）和（R）-BH4（3μM）中的羟基与6-甲基-PH4相比降低了Km。尽管发现6,6-二取代类似物的亲和力接近（R）-BH4（例如，6-丙基-6-甲基-PH4，4μM），但它们与磷酸化的TH的结合通常比其单取代对应物（6-苯基-PH4，0.8μM；参见6-苯基-6-甲基-PH4，8μM）更松散。提出了辅因子侧链结合结构域的模型，其中能够与（R）-BH4二羟丙基的烃主链进行范德华接触的有限区域的非极性蛋白质残基与羟基的识别位点相对。虽然与未磷酸化的酪氨酸羟化酶的亲水或疏水区域都可能发生相互作用，但cAMP依赖性蛋白激酶磷酸化似乎优化了这两种力的同时作用。
