大鼠胞外ADP-核糖基转移酶2.2的底物结合与催化作用

Substrate binding and catalysis of ecto-ADP-ribosyltransferase 2.2 from rat.

作者信息

Ritter Holger, Koch-Nolte Friedrich, Marquez Victor E, Schulz Georg E

机构信息

Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Albertstrasse 21, Freiburg im Breisgau 79104, Germany.

出版信息

Biochemistry. 2003 Sep 2;42(34):10155-62. doi: 10.1021/bi034625w.

DOI:10.1021/bi034625w

PMID:12939142

Abstract

The structures of beta-methylenethiazole-4-carboxamide adenine dinucleotide (TAD), NAD(+), and NADH as bound to ecto-ADP-ribosyltransferase 2.2 from rat and to its mutants E189I and E189A, respectively, have been established. The positions and conformations of NAD(+) and its analogues agree in general with those in other ADP-ribosyltransferases. The kinetic constants for NAD(+) hydrolysis were determined by RP-HPLC. The specific activity amounts to 26 units/mg, which is 6000-fold higher than a previously reported rate and 500-fold higher than the hydrolysis rates of other ADP-ribosyltransferases, confirming that hydrolysis is the major function of this enzyme. On the basis of structures and mutant activities, a catalytic mechanism is proposed. The known auto-ADP-ribosylation of the enzyme at the suggested position R184 is supported by one of the crystal structures where the nucleophile position is occupied by an Neta atom of this arginine which in turn is backed up by the base E159.

摘要

已确定β-亚甲基噻唑-4-甲酰胺腺嘌呤二核苷酸（TAD）、烟酰胺腺嘌呤二核苷酸（NAD⁺）和烟酰胺腺嘌呤二核苷酸还原型（NADH）与大鼠胞外ADP-核糖基转移酶2.2及其突变体E189I和E189A结合时的结构。NAD⁺及其类似物的位置和构象总体上与其他ADP-核糖基转移酶中的一致。通过反相高效液相色谱法（RP-HPLC）测定了NAD⁺水解的动力学常数。比活性为26单位/毫克，比先前报道的速率高6000倍，比其他ADP-核糖基转移酶的水解速率高500倍，证实水解是该酶的主要功能。基于结构和突变体活性，提出了一种催化机制。该酶在建议位置R184处已知的自动ADP-核糖基化得到了其中一个晶体结构的支持，在该结构中亲核试剂位置被该精氨酸的一个Nε原子占据，而该原子又得到碱基E159的支持。