Redox regulation of methionine aminopeptidase 2 activity.
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 Cys(228)-Cys(448) 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 Cys(228)-Cys(448) disulfide is at the rim of the active site and is only three residues distant from the catalytic His(231), 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.
From the Lowy Cancer Research Centre and Prince of Wales Clinical School, University of New South Wales, Sydney, New South Wales 2052, Australia.
Joyce
J
Chiu
From the Lowy Cancer Research Centre and Prince of Wales Clinical School, University of New South Wales, Sydney, New South Wales 2052, Australia.
Jason W H
JW
Wong
From the Lowy Cancer Research Centre and Prince of Wales Clinical School, University of New South Wales, Sydney, New South Wales 2052, Australia p.hogg@unsw.edu.au.
Philip J
PJ
Hogg
D000626:Aminopeptidases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D055162:Biocatalysis; D002460:Cell Line; D045744:Cell Line, Tumor; D003460:Crystallization; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005909:Glioblastoma / Q000201:enzymology / Q000473:pathology; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008666:Metalloendopeptidases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010455:Peptides / Q000378:metabolism; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D013879:Thioredoxins / Q000378:metabolism
D004220:Disulfides; D010455:Peptides; D011994:Recombinant Proteins; D013879:Thioredoxins; D000626:Aminopeptidases; C106541:methionine aminopeptidase 2; D008666:Metalloendopeptidases
Allosteric Regulation; Disulfide; Metalloprotease; Methionine; Oxidation-Reduction