Takishima K, Mamiya G
Department of Biochemistry, National Defense Medical College, Saitama, Japan.
Protein Seq Data Anal. 1987;1(2):103-6.
Jack bean urease is inactivated by the modification of about one cysteine residue per subunit with N-ethylmaleimide (NEM) or other thiol titrant. The location of this cysteine residue was identified. After blocking the unessential thiol groups with NEM, the essential cysteine was labeled with N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM). The DDPM-labeled protein was cleaved with cyanogen bromide and a DDPM-fragment was purified by gel filtration and ion exchange chromatography. Amino-terminal amino acid sequence of the DDPM-labeled fragment corresponded to that of a cyanogen bromide fragment of urease, VCHHLDREIPEDLAFAHSRIRKKTIAAEDVLNDIGAISIISSDSQAM. The second residue, Cys-592 in native urease, was labeled with DDPM. Fluoride ion, which competitively inhibits urease activity, protected urease from inactivation by NEM. The dissociation constant of fluoride ion obtained from the rate of inactivation with NEM was essentially identical to both the kinetically and spectrophotometrically determined dissociation constants. These suggest that the essential cysteine is at or near the active site.
刀豆脲酶可通过用N-乙基马来酰亚胺（NEM）或其他硫醇滴定剂修饰每个亚基约一个半胱氨酸残基而失活。确定了该半胱氨酸残基的位置。在用NEM封闭非必需硫醇基团后，用N-（4-二甲基氨基-3,5-二硝基苯基）马来酰亚胺（DDPM）标记必需的半胱氨酸。用溴化氰裂解DDPM标记的蛋白质，并通过凝胶过滤和离子交换色谱法纯化DDPM片段。DDPM标记片段的氨基末端氨基酸序列与脲酶的溴化氰片段的序列一致，即VCHHLDREIPEDLAFAHSRIRKKTIAAEDVLNDIGAISIISSDSQAM。天然脲酶中的第二个残基Cys-592被DDPM标记。竞争性抑制脲酶活性的氟离子可保护脲酶不被NEM失活。从NEM失活速率得到的氟离子解离常数与动力学和分光光度法测定的解离常数基本相同。这些结果表明必需的半胱氨酸位于活性位点或其附近。
