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Leukotriene A(4) hydrolase - Identification of a common carboxylate recognition site for the epoxide hydrolase and aminopeptidase substrates

Rudberg, P C; Tholander, F; Andberg, M; Thunnissen, Marjolein LU and Haeggstrom, J Z (2004) In Journal of Biological Chemistry 279(26). p.27376-27382
Abstract
Leukotriene ( LT) A(4) hydrolase is a bifunctional zinc metalloenzyme, which converts LTA(4) into the neutrophil chemoattractant LTB4 and also exhibits an anion-dependent aminopeptidase activity. In the x-ray crystal structure of LTA(4) hydrolase, Arg(563) and Lys(565) are found at the entrance of the active center. Here we report that replacement of Arg(563), but not Lys(565), leads to complete abrogation of the epoxide hydrolase activity. However, mutations of Arg(563) do not seem to affect substrate binding strength, because values of K-i for LTA(4) are almost identical for wild type and ( R563K) LTA(4) hydrolase. These results are supported by the 2.3-Angstrom crystal structure of (R563A) LTA(4) hydrolase, which does not reveal... (More)
Leukotriene ( LT) A(4) hydrolase is a bifunctional zinc metalloenzyme, which converts LTA(4) into the neutrophil chemoattractant LTB4 and also exhibits an anion-dependent aminopeptidase activity. In the x-ray crystal structure of LTA(4) hydrolase, Arg(563) and Lys(565) are found at the entrance of the active center. Here we report that replacement of Arg(563), but not Lys(565), leads to complete abrogation of the epoxide hydrolase activity. However, mutations of Arg(563) do not seem to affect substrate binding strength, because values of K-i for LTA(4) are almost identical for wild type and ( R563K) LTA(4) hydrolase. These results are supported by the 2.3-Angstrom crystal structure of (R563A) LTA(4) hydrolase, which does not reveal structural changes that can explain the complete loss of enzyme function. For the aminopeptidase reaction, mutations of Arg(563) reduce the catalytic activity (V-max = 0.3 - 20%), whereas mutations of Lys(565) have limited effect on catalysis (V-max = 58 - 108%). However, in (K565A)- and (K565M) LTA(4) hydrolase, i.e. mutants lacking a positive charge, values of the Michaelis constant for alanine-p-nitroanilide increase significantly (K-m = 480 - 640%). Together, our data indicate that Arg(563) plays an unexpected, critical role in the epoxide hydrolase reaction, presumably in the positioning of the carboxylate tail to ensure perfect substrate alignment along the catalytic elements of the active site. In the aminopeptidase reaction, Arg(563) and Lys(565) seem to cooperate to provide sufficient binding strength and productive alignment of the substrate. In conclusion, Arg(563) and Lys(565) possess distinct roles as carboxylate recognition sites for two chemically different substrates, each of which is turned over in separate enzymatic reactions catalyzed by LTA(4) hydrolase. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Biological Chemistry
volume
279
issue
26
pages
27376 - 27382
publisher
ASBMB
external identifiers
  • wos:000222120400068
  • scopus:3042549380
ISSN
1083-351X
DOI
10.1074/jbc.M401031200
language
English
LU publication?
yes
id
def7e5c1-7590-4fc3-beda-8a88e502f4e7 (old id 139393)
date added to LUP
2007-07-09 16:18:49
date last changed
2017-10-01 03:39:33
@article{def7e5c1-7590-4fc3-beda-8a88e502f4e7,
  abstract     = {Leukotriene ( LT) A(4) hydrolase is a bifunctional zinc metalloenzyme, which converts LTA(4) into the neutrophil chemoattractant LTB4 and also exhibits an anion-dependent aminopeptidase activity. In the x-ray crystal structure of LTA(4) hydrolase, Arg(563) and Lys(565) are found at the entrance of the active center. Here we report that replacement of Arg(563), but not Lys(565), leads to complete abrogation of the epoxide hydrolase activity. However, mutations of Arg(563) do not seem to affect substrate binding strength, because values of K-i for LTA(4) are almost identical for wild type and ( R563K) LTA(4) hydrolase. These results are supported by the 2.3-Angstrom crystal structure of (R563A) LTA(4) hydrolase, which does not reveal structural changes that can explain the complete loss of enzyme function. For the aminopeptidase reaction, mutations of Arg(563) reduce the catalytic activity (V-max = 0.3 - 20%), whereas mutations of Lys(565) have limited effect on catalysis (V-max = 58 - 108%). However, in (K565A)- and (K565M) LTA(4) hydrolase, i.e. mutants lacking a positive charge, values of the Michaelis constant for alanine-p-nitroanilide increase significantly (K-m = 480 - 640%). Together, our data indicate that Arg(563) plays an unexpected, critical role in the epoxide hydrolase reaction, presumably in the positioning of the carboxylate tail to ensure perfect substrate alignment along the catalytic elements of the active site. In the aminopeptidase reaction, Arg(563) and Lys(565) seem to cooperate to provide sufficient binding strength and productive alignment of the substrate. In conclusion, Arg(563) and Lys(565) possess distinct roles as carboxylate recognition sites for two chemically different substrates, each of which is turned over in separate enzymatic reactions catalyzed by LTA(4) hydrolase.},
  author       = {Rudberg, P C and Tholander, F and Andberg, M and Thunnissen, Marjolein and Haeggstrom, J Z},
  issn         = {1083-351X},
  language     = {eng},
  number       = {26},
  pages        = {27376--27382},
  publisher    = {ASBMB},
  series       = {Journal of Biological Chemistry},
  title        = {Leukotriene A(4) hydrolase - Identification of a common carboxylate recognition site for the epoxide hydrolase and aminopeptidase substrates},
  url          = {http://dx.doi.org/10.1074/jbc.M401031200},
  volume       = {279},
  year         = {2004},
}