Mechanism of the Reaction Catalyzed by Isoaspartyl Dipeptidase from Escherichia coli
详细信息 查看全文
- 作者：Ricardo Martí ; -Arbona ; Vicente Fresquet ; James B. Thoden ; Melissa L. Davis ; Hazel M. Holden ; and Frank M. Raushel
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：May 17, 2005
- 年：2005
- 卷：44
- 期：19
- 页码：7115 - 7124
- 全文大小：457K
- 年卷期：v.44,no.19(May 17, 2005)
- ISSN：1520-4995
文摘
Isoaspartyl dipeptidase (IAD) is a member of the amidohydrolase superfamily and catalyzesthe hydrolytic cleavage of 
-aspartyl dipeptides. Structural studies of the wild-type enzyme havedemonstrated that the active site consists of a binuclear metal center positioned at the C-terminal end ofa (/
)8-barrel domain. Steady-state kinetic parameters for the hydrolysis of -aspartyl dipeptides wereobtained at pH 8.1. The pH-rate profiles for the hydrolysis of -Asp-Leu were obtained for the Zn/Zn-,Co/Co-, Ni/Ni-, and Cd/Cd-substituted forms of IAD. Bell-shaped profiles were observed for kcat andkcat/Km as a function of pH for all four metal-substituted forms. The pKa of the group that must beunprotonated for catalytic activity varied according to the specific metal ion bound in the active site,whereas the pKa of the group that must be protonated for catalytic activity was relatively independent ofthe specific metal ion present. The identity of the group that must be unprotonated for catalytic activitywas consistent with the hydroxide that bridges the two divalent cations of the binuclear metal center. Theidentity of the group that must be protonated for activity was consistent with the free -amino group ofthe dipeptide substrate. Kinetic constants were obtained for the mutant enzymes at conserved residuesGlu77, Tyr137, Arg169, Arg233, Asp285, and Ser289. The catalytic properties of the wild-type and mutantenzymes, coupled with the X-ray crystal structure of the D285N mutant complexed with -Asp-His, areconsistent with a chemical reaction mechanism for the hydrolysis of dipeptides that is initiated by thepolarization of the amide bond via complexation to the -metal ion of the binuclear metal center.Nucleophilic attack by the bridging hydroxide is facilitated by abstraction of its proton by the side chaincarboxylate of Asp285. Collapse of the tetrahedral intermediate and cleavage of the carbon-nitrogenbond occur with donation of a proton from the protonated form of Asp285.
-aspartyl dipeptides. Structural studies of the wild-type enzyme havedemonstrated that the active site consists of a binuclear metal center positioned at the C-terminal end ofa (/)8-barrel domain. Steady-state kinetic parameters for the hydrolysis of -aspartyl dipeptides wereobtained at pH 8.1. The pH-rate profiles for the hydrolysis of -Asp-Leu were obtained for the Zn/Zn-,Co/Co-, Ni/Ni-, and Cd/Cd-substituted forms of IAD. Bell-shaped profiles were observed for kcat andkcat/Km as a function of pH for all four metal-substituted forms. The pKa of the group that must beunprotonated for catalytic activity varied according to the specific metal ion bound in the active site,whereas the pKa of the group that must be protonated for catalytic activity was relatively independent ofthe specific metal ion present. The identity of the group that must be unprotonated for catalytic activitywas consistent with the hydroxide that bridges the two divalent cations of the binuclear metal center. Theidentity of the group that must be protonated for activity was consistent with the free -amino group ofthe dipeptide substrate. Kinetic constants were obtained for the mutant enzymes at conserved residuesGlu77, Tyr137, Arg169, Arg233, Asp285, and Ser289. The catalytic properties of the wild-type and mutantenzymes, coupled with the X-ray crystal structure of the D285N mutant complexed with -Asp-His, areconsistent with a chemical reaction mechanism for the hydrolysis of dipeptides that is initiated by thepolarization of the amide bond via complexation to the -metal ion of the binuclear metal center.Nucleophilic attack by the bridging hydroxide is facilitated by abstraction of its proton by the side chaincarboxylate of Asp285. Collapse of the tetrahedral intermediate and cleavage of the carbon-nitrogenbond occur with donation of a proton from the protonated form of Asp285.