当前位置： SCI文献检索 > BIOCHEMISTRY期刊下所有文献 > Mechanistic studies of two dioxygenases in the methionine salvage pathway of Klebsiella pneumoniae.

Mechanistic studies of two dioxygenases in the methionine salvage pathway of Klebsiella pneumoniae.

Abstract：

:Two dioxygenases (ARD and ARD') were cloned from Klebsiella pneumoniae that catalyze different oxidative decomposition reactions of an advanced aci-reductone intermediate, CH(3)SCH(2)CH(2)COCH(OH)=CH(OH) (I), in the methionine salvage pathway. The two enzymes are remarkable in that they have the same polypeptide sequence but bind different metal ions (Ni(2+) and Fe(2+), respectively). ARD converts I to CH(3)SCH(2)CH(2)COOH, CO, and HCOOH. ARD' converts I to CH(3)SCH(2)CH(2)COCOOH and HCOOH. Kinetic analyses suggest that both ARD and ARD' have ordered sequential mechanisms. A model substrate (II), a dethio analogue of I, binds to the enzyme first as evidenced by its lambda(max) red shift upon binding. The dianion formation from II causes the same lambda(max) red shift, suggesting that II bind to the enzyme as a dianion. The electron-rich II dianion likely reacts with O(2) to form a peroxide anion intermediate. Previous (18)O(2) and (14)C tracer experiments established that ARD incorporates (18)O(2) into C(1) and C(3) of II and C(2) is released as CO. ARD' incorporates (18)O(2) into C(1) and C(2) of II. The product distribution seems to necessitate the formation of a five-membered cyclic peroxide intermediate for ARD and a four-membered cyclic peroxide intermediate for ARD'. A model chemical reaction demonstrates the chemical and kinetic competency of the proposed five-membered cyclic peroxide intermediate. The breakdown of the four-membered and five-membered cyclic peroxide intermediates gives the ARD' and ARD products, respectively. The nature of the metal ion appears to dictate the attack site of the peroxide anion and, consequently, the different cyclic peroxide intermediates and the different oxidative cleavages of II. A cyclopropyl substrate analogue inactivates both enzymes after multiple turnovers, providing evidence that a radical mechanism may be involved in the formation of the peroxide anion intermediate.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Dai Y,Pochapsky TC,Abeles RH

doi

10.1021/bi010110y

subject

Has Abstract

pub_date

2001-05-29 00:00:00

pages

6379-87

issue

eissn

0006-2960

issn

1520-4995

pii

bi010110y

journal_volume

pub_type

杂志文章

在线工具

Mechanistic studies of two dioxygenases in the methionine salvage pathway of Klebsiella pneumoniae.