当前位置： SCI文献检索 > BIOCHEMISTRY期刊下所有文献 > The catalytic mechanism of kynureninase from Pseudomonas fluorescens: evidence for transient quinonoid and ketimine intermediates from rapid-scanning stopped-flow spectrophotometry.

The catalytic mechanism of kynureninase from Pseudomonas fluorescens: evidence for transient quinonoid and ketimine intermediates from rapid-scanning stopped-flow spectrophotometry.

Abstract：

:The reaction of Pseudomonas fluorescens kynureninase with L-kynurenine and L-alanine has been examined using rapid-scanning stopped-flow spectrophotometry. Mixing kynureninase with 0.5 mM L-kynurenine results in formation of a quinonoid intermediate, with lambdamax = 494 nm, within the dead time (ca. 2 ms) of the stopped-flow mixer. This intermediate then decays rapidly, with k = 743 s-1, and this rate constant is reduced to 347 s-1 in [2H]H2O, suggesting that protonation of this intermediate by a solvent exchangeable proton takes place. Rapid quench experiments demonstrate that covalent changes in the cofactor occur, as pyridoxal 5'-phosphate is converted to pyridoxamine 5'-phosphate in about 30 mol % within 5 ms after mixing. Under single turnover conditions in the reaction of kynureninase with l-kynurenine, a transient shoulder absorbing at 335 nm is observed that may be a pyruvate ketimine intermediate. In contrast, the reaction of kynureninase with 0.5 mM l-kynurenine in the presence of 10 mM benzaldehyde results in the formation of a quinonoid intermediate (k = 67.4 s-1) with a very strong absorbance peak at 496 nm. The reaction of L-alanine with kynureninase exhibits the rapid formation (386 s-1 at 0.1 M) of an external aldimine intermediate absorbing at 420 nm, followed by slower formation of a quinonoid intermediate with a peak at 500 nm (k = 2.5 s-1). The 420 nm peak then decays slowly with concomitant formation of a peak at 320 nm corresponding to a pyruvate ketimine. These data demonstrate that quinonoid and ketimine intermediates are catalytically competent in the reaction mechanism of kynureninase, and provide additional support for our proposed mechanism for kynureninase from steady-state kinetic studies [Koushik, S. V., Sundararaju, B., and Phillips, R. S. Biochemistry 1998, 37, 1376-1382].

journal_name

Biochemistry

journal_title

Biochemistry

authors

Phillips RS,Sundararaju B,Koushik SV

doi

10.1021/bi980066v

subject

Has Abstract

pub_date

1998-06-16 00:00:00

pages

8783-9

issue

eissn

0006-2960

issn

1520-4995

pii

bi980066v

journal_volume

pub_type

杂志文章

在线工具

The catalytic mechanism of kynureninase from Pseudomonas fluorescens: evidence for transient quinonoid and ketimine intermediates from rapid-scanning stopped-flow spectrophotometry.