Characterization of the active site and insight into the binding mode of the anti-angiogenesis agent fumagillin to the manganese(II)-loaded methionyl aminopeptidase from Escherichia coli

Abstract

EPR spectra were recorded for methionine aminopeptidase from Escherichia coli (EcMetAP-I) samples (approximately 2.5 mM) to which one and two equivalents of Mn(II) were added (the latter is referred to as [MnMn(EcMetAP-I)]). The spectra for each sample were indistinguishable except that the spectrum of [MnMn(EcMetAP-I)] was twice as intense. The EPR spectrum of [MnMn(EcMetAP-I)] exhibited the characteristic six-line g approximately 2 EPR signal of mononuclear Mn(II) with A(av)((55)Mn)=9.3 mT (93 G) and exhibited Curie-law temperature dependence. This signal is typical of Mn(II) in a ligand sphere comprising oxygen and/or nitrogen atoms. Other features in the spectrum were observed only as the temperature was raised from that of liquid helium. The temperature dependences of these features are consistent with their assignment to excited state transitions in the S=1, 2 ... 5 non-Kramer's doublets, due to two antiferromagnetically coupled Mn(II) ions with an S=0 ground state. This assignment is supported by the observation of a characteristic 4.5 mT hyperfine pattern, and by the presence of signals in the parallel mode consistent with a non-Kramers' spin ladder. Upon the addition of the anti-angiogenesis agent fumagillin to [MnMn(EcMetAP-I)], very small changes were observed in the EPR spectrum. MALDI-TOF mass spectrometry indicated that fumagillin was, however, covalently coordinated to EcMetAP-I. Therefore, the inhibitory action of this anti-angiogenesis agent on EcMetAP-I appears to involve covalent binding to a polypeptide component at or near the active site rather than direct binding to the metal ions.

Structure of the angiogenesis inhibitor ovalicin bound to its noncognate target, human Type 1 methionine aminopeptidase

“…2B). The hydroxyl group on the inhibitor does not interact directly with either of the active site metal ions (closest distance 3.5 Å), consistent with EPR and EXAFS studies of Mn +2 ‐loaded Ec MetAP (D'Souza et al 2005). The inhibitor is surrounded by protein side chains and makes only a single backbone contact (a hydrogen bond to amide N–H of Cys301) (Fig.…”

“…This showed that the active site is accessible to these inhibitors but with lower affinity (Lowther et al 1998). Biochemical and spectroscopic studies (Lowther et al 1998; Cosper et al 2001; D'Souza et al 2005) have shed some light on the mode of binding of ovalicin to E. coli MetAP. At the same time the lack of a structure of the complex with Type 1 enzyme has limited the understanding of the difference in affinity between the Type 1 and the Type 2 enzymes.…”

Analyzing the catalytic role of Asp97 in the methionine aminopeptidase from Escherichia coli

“…Combination of these data suggests that mutating the only nonbridging ligand in the second divalent metal‐binding site in MetAPs to an alanine, which effectively removes the ability of the enzyme to form a dinuclear site, provides a MetAP enzyme that retains catalytic activity, albeit at extremely low levels. Reconciliation of these data with kinetic, ITC, crystallographic and EXAFS data suggesting that MetAPs are mononuclear with kinetic, MCD and EPR data indicating that metal binding is cooperative, at first glance, appears to be tricky [22,24,26,29,30]. However, the most logical explanation leads to the conclusion that metal binding to MetAPs is cooperative, and that discrepancies have arisen due to the concentrations of the enzyme samples used in the various experiments.…”

“…These data represent the first evidence that a dinuclear site can form in Ec MetAP‐I under physiological conditions. Moreover, EPR data recorded on Mn(II)‐loaded Ec MetAP‐I and Pf MetAP‐II suggest a small amount of dinuclear site formation after the addition of only a quarter equivalent of Mn(II) [22,29,30]. In order to determine whether a dinuclear site is required for enzymatic activity in MetAPs, the conserved aspartate, which is the lone nonbridging ligand for the M2 site in MetAPs (Fig.…”

Mutation of H63 and its catalytic affect on the methionine aminopeptidase from Escherichia coli

“…Under the conditions utilized, any cooperativity in divalent metal binding will not be detectable in ITC experiments due to high enzyme concentrations (∼70 μM) but may appear in EPR data. In fact, EPR data recorded on Mn(II)-loaded Ec MetAP-I and Pf MetAP-II suggests a small amount of dinuclear site formation even after the addition of only a quarter equivalent of Mn(II) [28,45,46]. Since activity titrations and ITC data are not particularly sensitive to the type of binding (i.e.…”