J 2003

Catalytic mechanism of the haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26

PROKOP, Zbyněk, Marta MONINCOVÁ, Radka CHALOUPKOVÁ, Martin KLVAŇA, Yuji NAGATA et. al.

Basic information

Original name

Catalytic mechanism of the haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26

Authors

PROKOP, Zbyněk (203 Czech Republic, guarantor), Marta MONINCOVÁ (203 Czech Republic), Radka CHALOUPKOVÁ (203 Czech Republic), Martin KLVAŇA (203 Czech Republic), Yuji NAGATA (392 Japan), Dick B. JANSSEN (528 Netherlands) and Jiří DAMBORSKÝ (203 Czech Republic)

Edition

Journal of Biological Chemistry, 2003, 0021-9258

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10600 1.6 Biological sciences

Country of publisher

United States of America

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

Impact factor

Impact factor: 6.482

RIV identification code

RIV/00216224:14310/03:00009155

Organization unit

Faculty of Science

UT WoS

000186452300008

Keywords in English

ENZYME; KINETICS; MECHANISM; LINB; HALOALKANE DEHALOGENASE

Tags

Změněno: 19/3/2010 11:05, prof. Mgr. Jiří Damborský, Dr.

Abstract

V originále

Haloalkane dehalogenases are bacterial enzymes capable of carbon-halogen bond cleavage in halogenated compounds. To obtain insight in the mechanism of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB), we studied the steady-state and pre-steady-state kinetics of the conversion of the substrates 1-chlorohexane, chlorocyclohexane and bromocyclohexane. The results lead to a proposal of a minimal kinetic mechanism consisting of three main steps: (i) substrate binding, (ii) cleavage of the carbon-halogen bond with simultaneous formation of an alkyl-enzyme intermediate and (iii) hydrolysis of the alkyl-enzyme intermediate. Release of both products, halide and alcohol, is a fast process that was not included in reaction mechanism as a distinct step. Comparison of the kinetic mechanism of LinB with that of haloalkane dehalogenase DhlA from Xantobacter autotrophicus GJ10 and the haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 shows that the overall mechanisms are similar. The main difference is in the rate-limiting step, which is hydrolysis of the alkyl-enzyme intermediate in LinB, halide release in DhlA, and liberation of an alcohol in DhaA. The occurrence of different rate-limiting steps for three enzymes that belong to the same protein family indicates that extrapolation of this important catalytic property from one enzyme to another can be misleading even for evolutionary closely related proteins. The differences in the rate-limiting step were related to: (i) a number and size of the entrance tunnels, (ii) protein flexibility and (iii) composition of the halide-stabilizing active site residues based on comparison of protein structures.

Links

LN00A016, research and development project
Name: BIOMOLEKULÁRNÍ CENTRUM
Investor: Ministry of Education, Youth and Sports of the CR, Biomolecular Center