Molecular Discrimination of Type-I over Type-II Methionyl Aminopeptidases

• Published in: Biochemistry (Easton) Vol. 44; no. 36; pp. 12049 - 12056
• Main Authors: Swierczek, Krzysztof, Copik, Alicja J, Swierczek, Sabina I, Holz, Richard C
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 13.09.2005
• Subjects: Amino Acid Sequence; Aminopeptidases - chemistry; Aminopeptidases - classification; Aminopeptidases - genetics; Aminopeptidases - metabolism; Anti-Infective Agents - pharmacology; Cysteine - genetics; Cysteine - metabolism; Escherichia coli - enzymology; Humans; Kinetics; Methionyl Aminopeptidases; Molecular Sequence Data; Mutation - genetics; Pyrococcus furiosus - enzymology; Sequence Alignment; Sequence Homology, Amino Acid; Species Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi047752g

Two residues that are conserved in type-I methionyl aminopeptidases (MetAPs) but are absent in all type-II MetAPs are the cysteine residues (Escherichia coli MetAP-I:  C59 and C70) that reside at the back of the substrate recognition pocket. These Cys residues are 4.4 Å apart and do not form a disulfide bond. Since bacteria and fungi contain only type-I MetAPs while all human cells contain both type-I and type-II MetAPs, type-I MetAPs represent a novel antibiotic/antifungal target if type-I MetAPs can be specifically targeted over type-II. Based on reaction of the thiol-specific binding reagent 5,5‘-dithio-bis(2-nitrobenzoic acid) (DTNB) with the type-I MetAP from E. coli and the type-II MetAP from Pyrococcus furiosus, the type-I MetAP can be selectively inhibited. Verification that DTNB covalently binds to C59 in EcMetAP-I was obtained by mass spectrometry (MS) from reaction of DTNB with the C59A and C70A mutant EcMetAP-I enzymes. In addition, two inhibitors of EcMetAP-I, 5-iodopentaphosphonic acid (1) and 6-phosphonohexanoic acid (2), were designed and synthesized. The first was designed as a selective-C59 binding reagent while the second was designed as a simple competitive inhibitor of EcMetAP. Indeed, inhibitor 1 forms a covalent interaction with C59 based on activity assays and MS measurements, while 2 does not. These data indicate that type-I MetAPs can be selectively targeted over type-II MetAPs, suggesting that type-I MetAPs represent a new enzymatic target for antibacterial or antifungal agents.