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PMID:15078870

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Citation

Rudberg, PC, Tholander, F, Andberg, M, Thunnissen, MM and Haeggström, JZ (2004) Leukotriene A4 hydrolase: identification of a common carboxylate recognition site for the epoxide hydrolase and aminopeptidase substrates. J. Biol. Chem. 279:27376-82

Abstract

Leukotriene (LT) A(4) hydrolase is a bifunctional zinc metalloenzyme, which converts LTA(4) into the neutrophil chemoattractant LTB(4) 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-A 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.

Links

PubMed Online version:10.1074/jbc.M401031200

Keywords

Amino Acid Sequence; Aminopeptidases/metabolism; Arginine/genetics; Arginine/metabolism; Binding Sites; Carboxylic Acids/metabolism; Catalysis; Crystallography, X-Ray; Enzyme Inhibitors/pharmacology; Epoxide Hydrolases/antagonists & inhibitors; Epoxide Hydrolases/chemistry; Epoxide Hydrolases/genetics; Epoxide Hydrolases/metabolism; Escherichia coli/metabolism; Humans; Hydroxamic Acids/pharmacology; Leukotriene A4/pharmacology; Lysine/genetics; Lysine/metabolism; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Sequence Alignment; Static Electricity

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:LKHA4

enables

GO:0004177: aminopeptidase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:LKHA4

enables

GO:0004301: epoxide hydrolase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:LKHA4

enables

GO:0004463: leukotriene-A4 hydrolase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:LKHA4

enables

GO:0008270: zinc ion binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:LKHA4

involved_in

GO:0019370: leukotriene biosynthetic process

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:LKHA4

involved_in

GO:0043171: peptide catabolic process

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete


See also

References

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