Robertson J G, Kumar A, Mancewicz J A, Villafranca J J
Department of Chemistry, Pennsylvania State University, University Park 16802.
J Biol Chem. 1989 Nov 25;264(33):19916-21.
Bovine dopamine beta-hydroxylase was examined spectroscopically for the presence of covalently bound pyrroloquinoline quinone (PQQ). Pure dopamine beta-hydroxylase had a featureless UV-visible spectrum above 300 nm. An equimolar solution of dopamine beta-hydroxylase and exogenously added PQQ (1 PQQ/active site) had a strong absorption maximum at 333 nm. Dialysis removed the added PQQ, indicating that dopamine beta-hydroxylase does not bind PQQ irreversibly. Reaction of dopamine beta-hydroxylase with 6 mM phenylhydrazine in the presence of 15 mM ascorbate caused 96% inactivation within 20 min and did not produce any spectrally detectable amounts of the phenylhydrazone adduct of PQQ, as reported by van der Meer et al. (van der Meer, R.A., Jongejan, J.A., and Duine, J.A. (1988) FEBS Lett. 231, 303-307). The peptide profile of phenylhydrazine inactivated dopamine beta-hydroxylase was monitored at 316 nm and did not reveal any peptides that might contain a PQQ-phenylhydrazone adduct. Thus, the absence of any spectrally detectable PQQ-phenylhydrazone adducts under these conditions demonstrates that the mechanism of phenylhydrazine inactivation does not involve covalent modification of PQQ at the active site of dopamine beta-hydroxylase and provides strong evidence that the native enzyme does not contain PQQ.
采用光谱法检测牛多巴胺β-羟化酶中是否存在共价结合的吡咯喹啉醌（PQQ）。纯多巴胺β-羟化酶在300nm以上具有无特征的紫外可见光谱。多巴胺β-羟化酶与外源添加的PQQ（1个PQQ/活性位点）的等摩尔溶液在333nm处有一个强吸收峰。透析可除去添加的PQQ，这表明多巴胺β-羟化酶不会不可逆地结合PQQ。如van der Meer等人所报道（van der Meer, R.A., Jongejan, J.A., and Duine, J.A. (1988) FEBS Lett. 231, 303 - 307），多巴胺β-羟化酶在15mM抗坏血酸存在下与6mM苯肼反应，20分钟内导致酶失活96%，且未产生任何光谱可检测量的PQQ苯腙加合物。在316nm处监测苯肼使多巴胺β-羟化酶失活后的肽谱，未发现任何可能含有PQQ苯腙加合物的肽段。因此，在这些条件下未检测到任何光谱可检测的PQQ苯腙加合物，表明苯肼失活机制不涉及多巴胺β-羟化酶活性位点处PQQ的共价修饰，并有力证明天然酶不含PQQ。
