Redox Regulation of Methionine Aminopeptidase 2 Activity

• Published in: The Journal of biological chemistry 2014-05, Vol.289 (21), p.15035-15043
• Main Authors: Chiu, Joyce, Wong, Jason W.H., Hogg, Philip J.
• Format: Article
• Language: English
• Subjects: Allosteric Regulation; Aminopeptidases - classification; Aminopeptidases - genetics; Aminopeptidases - metabolism; Animals; Biocatalysis; Cell Line; Cell Line, Tumor; Crystallization; Disulfide; Disulfides - chemistry; Disulfides - metabolism; Electrophoresis, Polyacrylamide Gel; Enzymology; Glioblastoma - enzymology; Glioblastoma - pathology; Humans; Hydrolysis; Kinetics; Metalloendopeptidases - classification; Metalloendopeptidases - genetics; Metalloendopeptidases - metabolism; Metalloprotease; Methionine; Models, Molecular; Oxidation-Reduction; Peptides - metabolism; Phylogeny; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Substrate Specificity; Tandem Mass Spectrometry; Thioredoxins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.554253

Protein translation is initiated with methionine in eukaryotes, and the majority of proteins have their N-terminal methionine removed by methionine aminopeptidases (MetAP1 and MetAP2) prior to action. Methionine removal can be important for protein function, localization, or stability. No mechanism of regulation of MetAP activity has been identified. MetAP2, but not MetAP1, contains a single Cys228-Cys448 disulfide bond that has an −RHStaple configuration and links two β-loop structures, which are hallmarks of allosteric disulfide bonds. From analysis of crystal structures and using mass spectrometry and activity assays, we found that the disulfide bond exists in oxidized and reduced states in the recombinant enzyme. The disulfide has a standard redox potential of −261 mV and is efficiently reduced by the protein reductant, thioredoxin, with a rate constant of 16,180 m−1 s−1. The MetAP2 disulfide bond also exists in oxidized and reduced states in glioblastoma tumor cells, and stressing the cells by oxygen or glucose deprivation results in more oxidized enzyme. The Cys228-Cys448 disulfide is at the rim of the active site and is only three residues distant from the catalytic His231, which suggested that cleavage of the bond would influence substrate hydrolysis. Indeed, oxidized and reduced isoforms have different catalytic efficiencies for hydrolysis of MetAP2 peptide substrates. These findings indicate that MetAP2 is post-translationally regulated by an allosteric disulfide bond, which controls substrate specificity and catalytic efficiency. The N-terminal methionine in new eukaryote proteins is removed by methionine aminopeptidases, but how these enzymes are regulated is not known. Methionine aminopeptidase 2 contains a single disulfide bond that exists in oxidized and reduced states and influences enzyme function. MetAP2 is regulated by an allosteric disulfide bond. This has implications for MetAP2 substrate proteins and other similar enzymes.