Naish-Byfield S, Cooksey C J, Riley P A
Department of Biology and Biochemistry, Brunel University, Uxbridge, Middx, U.K.
Biochem J. 1994 Nov 15;304 ( Pt 1)(Pt 1):155-62. doi: 10.1042/bj3040155.
The effect of thiol compounds on the monophenolase activity of tyrosinase was investigated using 4-hydroxyanisole as the substrate and dithiothreitol (DTT) as the model thiol compound. We have demonstrated three actions of DTT on tyrosinase-catalysed reactions: (1) direct reduction of the copper at the active site of the enzyme; (2) generation of secondary, oxidizable species by adduct formation with the o-quinone reaction product, 4-MOB, which leads to an increase in the total oxygen utilization by the reaction system; and (3) reversible inhibition of the enzyme. We confirm our previous observation that, at approx. 10 mol of DTT/mol of enzyme, the lag phase associated with monohydric phenol oxidation by tyrosinase is abolished. We suggest that this is due to reduction of the copper at the active site of the enzyme by DTT, since (a) reduction of active-site copper in situ by DTT was demonstrated by [Cu(I)]2-carbon monoxide complex formation and (b) abolition of the lag at low DTT concentration occurs without effect on the maximum rate of reaction or on the total amount of oxygen utilized. At concentrations of DTT above that required to abolish the lag, we found that the initial velocity of the reaction increased with increasing DTT, with a concomitant increase in the total oxygen utilization. This is due to the formation of DTT-4-methoxy-o-benzoquinone (4-MOB) adducts which provide additional dihydric phenol substrate either directly or by reducing nascent 4-MOB. We present n.m.r. evidence for the formation of mono- and di-aromatic DTT adducts with 4-MOB, consistent with a suggested reoxidation scheme in the presence of tyrosinase. Inhibition of the enzyme at concentrations of DTT above 300 pmol/unit of enzyme was released on exhaustion of DTT by adduct formation with 4-MOB as it was generated.
以4-羟基苯甲醚为底物，二硫苏糖醇（DTT）为模型硫醇化合物，研究了硫醇化合物对酪氨酸酶单酚酶活性的影响。我们证明了DTT对酪氨酸酶催化反应的三种作用：（1）直接还原酶活性位点的铜；（2）与邻醌反应产物4-甲氧基苯醌（4-MOB）形成加合物，生成可氧化的次级物种，导致反应体系总氧利用率增加；（3）对酶的可逆抑制。我们证实了之前的观察结果，即在大约10摩尔DTT/摩尔酶时，酪氨酸酶氧化一元酚相关的延迟期被消除。我们认为这是由于DTT还原了酶活性位点的铜，因为（a）通过[Cu（I）]2-一氧化碳络合物的形成证明了DTT原位还原活性位点的铜，并且（b）在低DTT浓度下延迟的消除对最大反应速率或总氧利用量没有影响。在高于消除延迟所需的DTT浓度下，我们发现反应的初始速度随着DTT的增加而增加，同时总氧利用率也随之增加。这是由于形成了DTT-4-甲氧基邻苯醌（4-MOB）加合物，这些加合物直接或通过还原新生的4-MOB提供了额外的二元酚底物。我们提供了核磁共振证据，证明了与4-MOB形成单芳族和二芳族DTT加合物，这与酪氨酸酶存在下的建议再氧化方案一致。当DTT与生成的4-MOB形成加合物而耗尽时，高于300 pmol/酶单位的DTT浓度对酶的抑制作用被解除。
