Srivastava PK, Kayastha AM
School of Biotechnology, Faculty of Science, Banaras Hindu University, 221 005, Varanasi, India
Plant Sci. 2000 Oct 16;159(1):149-158. doi: 10.1016/s0168-9452(00)00343-5.
Titration of urease from pigeonpea (Cajanus cajan L.), a hexameric protein (mol. wt. 480000; subunit mol. wt. 80000), with 5,5'-dithiobis-(2-nitrobenzoate) (DTNB) reveals the presence of 5.82+/-0.13 'accessible' sulfhydryl groups per molecule of the enzyme protein (i.e. about one 'accessible' SH group per subunit). Denatured enzyme was found to titrate for 12.1+/-0.1 SH groups per molecule (i.e. about two SH groups per subunit). Half of the 'accessible' groups react faster than the remaining at pH 8.5 as well as pH 7.5. However, the reaction was slower at pH 7.5 than 8.5. Time-dependent loss of enzyme activity with DTNB was also found to be biphasic. The enzyme was inactivated at low concentration of p-chloromercuribenzoate (p-CMB), N-ethyl maleimide (NEM) and iodoacetamide. The inactivation reactions were biphasic, with half of the activity lost more rapidly than the remaining half. The loss of activity with p-CMB was linearly related to the blocking of accessible SH groups. Inactivation by p-CMB is largely reversible by addition of excess of cysteine. Fluoride ion strongly protects the enzyme against NEM inactivation, however, substrate urea provides much weaker protection against SH group reagents. The significance of these results is discussed.
用5,5'-二硫代双（2-硝基苯甲酸）（DTNB）对来自木豆（Cajanus cajan L.）的脲酶进行滴定，该脲酶是一种六聚体蛋白（分子量480000；亚基分子量80000），结果显示每分子酶蛋白中存在5.82±0.13个“可及”巯基（即每个亚基约有一个“可及”的SH基团）。发现变性酶每分子可滴定12.1±0.1个SH基团（即每个亚基约有两个SH基团）。在pH 8.5和pH 7.5时，一半的“可及”基团反应速度比其余基团快。然而，在pH 7.5时的反应比pH 8.5时慢。还发现酶活性随DTNB的时间依赖性丧失是双相的。该酶在低浓度的对氯汞苯甲酸（p-CMB）、N-乙基马来酰亚胺（NEM）和碘乙酰胺作用下失活。失活反应是双相的，一半活性的丧失比另一半更快。p-CMB导致的活性丧失与可及SH基团的封闭呈线性相关。加入过量的半胱氨酸后，p-CMB导致的失活在很大程度上是可逆的。氟离子能强烈保护该酶免受NEM失活的影响，然而，底物尿素对SH基团试剂的保护作用要弱得多。讨论了这些结果的意义。
