Mattsson P, Pohjalainen T, Korpela T
Department of Biochemistry, University of Turku, Finland.
Biochim Biophys Acta. 1992 Jul 13;1122(1):33-40. doi: 10.1016/0167-4838(92)90123-u.
Counting of integral numbers of cysteine residues of the reduced and denaturated form of cyclomaltodextrin glucanotransferase (CGTase) from Bacillus circulans var. alkalophilus (ATCC 21783) showed two cysteine residues per enzyme molecule. Titrations of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) led to the same result. No free SH-group was detected in denatured form of CGTase, indicating that the two cysteine residues are linked by one disulfide bridge. Cyclizing activity of the GdmCl-denaturated and reduced enzyme was 13% of that of the native one. Incubation of CGTase with diethylpyrocarbonate (DEP) showed a pseudo-first-order inhibition with second-order rate constant of 3.2 M-1 s-1. Reaction with hydroxylamine and spectroscopic studies implied that inactivation of CGTase by DEP is due to modification of one histidine residue concomitantly with a 50% decrease in the cyclizing activity (t1/2 = 10.8 min). The inhibition was partially reversible. CGTase was protected against inactivation by alpha- and beta-cyclodextrins suggesting that the modified histidine residue is at or near the active site. Conversion of starch with DEP-modified enzyme resulted in a decreased formation of cyclodextrins while the relative amount of reducing sugars increased. Preliminary results on modification of CGTase with other reagents, e.g., Woodward's reagent K, 2,3-butanedione and carbodiimide are included.
对嗜碱环状芽孢杆菌（ATCC 21783）的环糊精葡聚糖转移酶（CGTase）还原和变性形式的半胱氨酸残基整数进行计数，结果显示每个酶分子有两个半胱氨酸残基。用5,5'-二硫代双（2-硝基苯甲酸）对该酶进行滴定也得到了相同的结果。在变性的CGTase中未检测到游离的巯基，这表明这两个半胱氨酸残基通过一个二硫键相连。盐酸胍变性并还原的酶的环化活性为天然酶的13%。用焦碳酸二乙酯（DEP）处理CGTase显示出假一级抑制，二级速率常数为3.2 M-1 s-1。与羟胺的反应和光谱研究表明，DEP使CGTase失活是由于一个组氨酸残基的修饰，同时环化活性降低了50%（t1/2 = 10.8分钟）。这种抑制作用部分是可逆的。α-和β-环糊精可保护CGTase不被失活，这表明被修饰的组氨酸残基位于活性位点或其附近。用DEP修饰的酶转化淀粉会导致环糊精的生成减少，而还原糖的相对量增加。文中还包括了用其他试剂，如伍德沃德试剂K、2,3-丁二酮和碳二亚胺对CGTase进行修饰的初步结果。
