Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase,
详细信息    查看全文
文摘
Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (kcat = 2.0 s鈭?; kcat/Km = 2.5 脳 103 M鈭? s鈭?). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn2+ prior to induction, the purified enzyme was substantially more active for the deamination of adenine with kcat and kcat/Km values of 200 s鈭? and 5 脳 105 M鈭? s鈭?, respectively. The apoenzyme was prepared and reconstituted with Fe2+, Zn2+, or Mn2+. In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [FeII/FeII]-ADE was oxidized to [FeIII/FeIII]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [FeIII/FeIII]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or M枚ssbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 脜 resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine鈭扐DE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH鈭抮ate profiles, and solvent viscosity were utilized to propose a chemical reaction mechanism and the identity of the rate-limiting steps.

© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号

地址:北京市海淀区学院路29号 邮编:100083

电话:办公室:(+86 10)66554848;文献借阅、咨询服务、科技查新:66554700