Mutations at the S1 sites of methionine aminopeptidases from Escherichia coli and Homo sapiens reveal the residues critical for substrate specificity

Methionine aminopeptidase (MetAP) catalyzes the removal of methionine from newly synthesized polypeptides. MetAP carries out this cleavage with high precision, and Met is the only natural amino acid residue at the N terminus that is accepted, although type I and type II MetAPs use two different sets of residues to form the hydrophobic S1 site. Characteristics of the S1 binding pocket in type I MetAP were investigated by systematic mutation of each of the seven S1 residues in Escherichia coli MetAP type I (EcMetAP1) and human MetAP type I (HsMetAP1). We found that Tyr-65 and Trp-221 in EcMetAP1, as well as the corresponding residues Phe-197 and Trp-352 in HsMetAP1, were essential for the hydrolysis of a thiopeptolide substrate, Met-S-Gly-Phe. Mutation of Phe-191 to Ala in HsMetAP1 caused inactivity in contrast to the full activity of EcMetAP1(Y62A), which may suggest a subtle difference between the two type I enzymes. The more striking finding is that mutation of Cys-70 in EcMetAP1 or Cys-202 in HsMetAP1 opens up the S1 pocket. The thiopeptolides Leu-S-Gly-Phe and Phe-S-Gly-Phe, with previously unacceptable Leu or Phe as the N-terminal residue, became efficient substrates of EcMetAP1(C70A) and HsMetAP1(C202A). The relaxed specificity shown in these S1 site mutants for the N-terminal residues was confirmed by hydrolysis of peptide substrates and inhibition by reaction products. The structural features at the enzyme active site will be useful information for designing specific MetAP inhibitors for therapeutic applications.     

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

Methionine aminopeptidases (MetAPs) remove the initiator methionine during protein biosynthesis. They exist in two isoforms, MetAP1 and MetAP2. The anti-angiogenic compound fumagillin binds tightly to the Type 2 MetAPs but only weakly to Type 1. High-affinity complexes of fumagillin and its relative ovalicin with Type 2 human MetAP have been reported. Here we describe the crystallographic structure of the low-affinity complex between ovalicin and Type 1 human MetAP at 1.1 A resolution. This provides the first opportunity to compare the structures of ovalicin or fumagillin bound to a Type 1 and a Type 2 MetAP. For both Type 1 and Type 2 human MetAPs the inhibitor makes a covalent adduct with a corresponding histidine. At the same time there are significant differences in the alignment of the inhibitors within the respective active sites. It has been argued that the lower affinity of ovalicin and fumagillin for the Type 1 MetAPs is due to the smaller size of their active sites relative to the Type 2 enzymes. Comparison with the uncomplexed structure of human Type 1 MetAP indicates that there is some truth to this. Several active site residues have to move "outward" by 0.5 Angstroms or so to accommodate the inhibitor. Other residues move "inward." There are, however, other factors that come into play. In particular, the side chain of His310 rotates by 134 degrees into a different position where (together with Glu128 and Tyr195) it coordinates a metal ion not seen at this site in the native enzyme.     

Analysis of the interplay of protein biogenesis factors at the ribosome exit site reveals new role for NAC

The ribosome exit site is a focal point for the interaction of protein-biogenesis factors that guide the fate of nascent polypeptides. These factors include chaperones such as NAC, N-terminal-modifying enzymes like Methionine aminopeptidase (MetAP), and the signal recognition particle (SRP), which targets secretory and membrane proteins to the ER. These factors potentially compete with one another in the short time-window when the nascent chain first emerges at the exit site, suggesting a need for regulation. Here, we show that MetAP contacts the ribosome at the universal adaptor site where it is adjacent to the α subunit of NAC. SRP is also known to contact the ribosome at this site. In the absence of NAC, MetAP and SRP antagonize each other, indicating a novel role for NAC in regulating the access of MetAP and SRP to the ribosome. NAC also functions in SRP-dependent targeting and helps to protect substrates from aggregation before translocation.     

Kinetic and mutational studies of the number of interacting divalent cations required by bacterial and human methionine aminopeptidases

Methionine aminopeptidases (MetAP) are responsible for the proteolytic removal of the initiator methionine from nascent proteins. This processing permits multiple posttranslational modifications and protein turnover. We have cloned, expressed in Escherichia coli, and purified the recombinant human mitochondrial MetAP isoform (MetAP1D). The full-length enzyme and a truncated form lacking the mitochondrial targeting sequence (residues 1-55) have been characterized as metal-requiring proteases, with Co2+ being the best activator. At the optimal pH (8.0), the kcat of MetAP1D of 0.39 min-1 is 280-fold lower, and the Km of the substrate Met-Pro-p-nitroanilide (576 microM) is 3-fold greater, than the respective kinetic parameters obtained with MetAP from E. coli, although MetAP1D is 61% homologous to E. coli MetAP and their circular dichroic spectra are nearly identical. MetAP1D thus appears to be a less efficient enzyme than other known MetAPs in vitro. At saturating substrate concentrations, a plot of Vmax versus free Co2+ shows sigmoidal metal activation of MetAP1D, both with and without an N-terminal His-tag, with a Hill coefficient (n) of 1.9 and a K0.5 of 0.40 microM. Similarly, E. coli MetAP shows n = 2.1 and K0.5 = 0.2 microM. Hence, at least two Co2+ ions, which may act cooperatively, are needed to promote catalysis, providing kinetic evidence for the functioning of both Co2+ ions of the binuclear complex found in the X-ray structure of E. coli MetAP [Roderick, S. L. and Matthews, B. W. (1993) Biochemistry 32, 3907-3912] and resolving a disagreement in the literature. The X-ray structure of the human cytosolic MetAP1 showed three Co2+ ions at the active site, with the third Co2+ coordinated by the conserved residue His 212 [Addlagatta, A., Hu, X., Liu, J. O., and Matthews, B. W. (2005) Biochemistry 44, 14741-14749]. Consistent with the structure, kinetic studies of the human cytosolic MetAP1 yielded a Hill coefficient (n) of 2.9 and a K0.5 of 0.26 microM for activation by Co2+, as well as a kcat of 25.5 min-1 and a Km of 740 microM for the substrate Met-Pro-p-nitroanilide. The H212A mutation decreased n to 2.2, decreased kcat 60-fold to 0.42 min-1, and increased K0.5 6.5-fold to 1.8 microM. The H212K mutation further decreased n to 1.4, decreased kcat 1800-fold to 0.014 min-1, and increased K0.5 158-fold to 41 microM. Hence, at least three Co2+ ions are needed to promote optimal catalysis by human MetAP1. Both mutations of His212 abolished the binding and/or the cooperativity of the third Co2+ ion, as indicated by the decreases in n and the increases in K0.5 of the remaining two Co2+ ions, but did not affect the Km of the substrate. The more damaging effects of the H212K mutation on both the Hill coefficient for Co2+ binding and the catalysis suggest that Lys 212 might directly compete with Co2+ for the third metal-binding site. Together, these results suggest that human MetAP1 is distinct from other members of the MetAP superfamily in the number of metal ions employed and likely mechanism of catalysis.     

Structural basis for the functional differences between type I and type II human methionine aminopeptidases

Determination of the crystal structure of human MetAP1 makes it possible, for the first time, to compare the structures of a Type I and a Type II methionine aminopeptidase (MetAP) from the same organism. Comparison of the Type I enzyme with the previously reported complex of ovalicin with Type II MetAP shows that the active site of the former is reduced in size and would incur steric clashes with the bound inhibitor. This explains why ovalicin and related anti-angiogenesis inhibitors target Type II human MetAP but not Type I. The differences in both size and shape of the active sites between MetAP1 and MetAP2 also help to explain their different substrate specificity. In the presence of excess Co(2+), a third cobalt ion binds in the active site region, explaining why metal ions in excess can be inhibitory. Also, the N-terminal region of the protein contains three distinct Pro-x-x-Pro motifs, supporting the prior suggestion that this region of the protein may participate in binding to the ribosome.     

Divalent metal binding properties of the methionyl aminopeptidase from Escherichia coli

The metal-binding properties of the methionyl aminopeptidase from Escherichia coli (MetAP) were investigated. Measurements of catalytic activity as a function of added Co(II) and Fe(II) revealed that maximal enzymatic activity is observed after the addition of only 1 equiv of divalent metal ion. Based on these studies, metal binding constants for the first metal binding event were found to be 0.3 +/- 0.2 microM and 0.2 +/- 0.2 microM for Co(II)- and Fe(II)-substituted MetAP, respectively. Binding of excess metal ions (>50 equiv) resulted in the loss of approximately 50% of the catalytic activity. Electronic absorption spectral titration of a 1 mM sample of MetAP with Co(II) provided a binding constant of 2.5 +/- 0.5 mM for the second metal binding site. Furthermore, the electronic absorption spectra of Co(II)-loaded MetAP indicated that both metal ions reside in a pentacoordinate geometry. Consistent with the absorption data, electron paramagnetic resonance (EPR) spectra of [CoCo(MetAP)] also indicated that the Co(II) geometries are not highly constrained, suggesting that each Co(II) ion in MetAP resides in a pentacoordinate geometry. EPR studies on [CoCo(MetAP)] also revealed that at pH 7.5 there is no significant spin-coupling between the two Co(II) ions, though a small proportion ( approximately 5%) of the sample exhibited detectable spin-spin interactions at pH values > 9.6. EPR studies on [Fe(III)_(MetAP)] and [Fe(III)Fe(III)(MetAP)] also suggested no spin-coupling between the two metal ions. (1)H nuclear magnetic resonance (NMR) spectra of [Co(II)_(MetAP)] in both H(2)O and D(2)O buffer indicated that the first metal binding site contains the only active-site histidine residue, His171. Mechanistic implications of the observed binding properties of divalent metal ions to the MetAP from E. coli are discussed.     

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 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.     

Yeast methionine aminopeptidase type 1 is ribosome-associated and requires its N-terminal zinc finger domain for normal function in vivo

Methionine aminopeptidase type 1 (MetAP1) cotranslationally removes N-terminal methionine from nascent polypeptides, when the second residue in the primary structure is small and uncharged. Eukaryotic MetAP1 has an N-terminal zinc finger domain not found in prokaryotic MetAPs. We hypothesized that the zinc finger domain mediates the association of MetAP1 with the ribosomes and have reported genetic evidence that it is important for the normal function of MetAP1 in vivo. In this study, the intracellular role of the zinc finger domain in yeast MetAP1 function was examined. Wild-type MetAP1 expressed in a yeast map1 null strain removed 100% of N-terminal methionine from a reporter protein, while zinc finger mutants removed only 31-35%. Ribosome profiles of map1 null expressing wild-type MetAP1 or one of three zinc finger mutants were compared. Wild-type MetAP1 was found to be an 80S translational complex-associated protein that primarily associates with the 60S subunit. Deletion of the zinc finger domain did not significantly alter the ribosome profile distribution of MetAP1. In contrast, single point mutations in the first or second zinc finger motif disrupted association of MetAP1 with the 60S subunit and the 80S translational complex. Together, these results indicate that the zinc finger domain is essential for the normal processing function of MetAP1 in vivo and suggest that it may be important for the proper functional alignment of MetAP1 on the ribosomes.     

Functional expression of human methionine aminopeptidase type 1 in Saccharomyces cerevisiae

We expressed recombinant human methionine aminopeptidase type 1 (MAP or MetAP) in a map1 null yeast strain to determine the extent of functional complementation between the two proteins. The human MetAP1 protein fully rescued the slow growth phenotype associated with deletion of yeast MetAP1, suggesting that the yeast and human MetAP1 proteins may have similar roles in vivo. Expression of human MetAP1 in yeast has significance in understanding the function of the human protein, studying its in vivo substrate specificity, and developing specific anti-fungal drugs to target yeast MetAP1.     

A single amino acid difference between archaeal and human type 2 methionine aminopeptidases differentiates their affinity towards ovalicin

In almost all living cells, methionine aminopeptidase (MetAP) co-translationally cleaves the initiator methionine in at least 70% of the newly synthesized polypeptides. MetAPs are typically classified into Type 1 and Type 2. While prokaryotes and archaea contain only either Type 1 or Type 2 MetAPs respectively, eukaryotes contain both types of enzymes. Almost all MetAPs published till date cleave only methionine from the amino terminus of the substrate peptides. Earlier experiments on crude Type 2a MetAP isolated from Pyrococcus furiosus (PfuMetAP2a) cosmid protein library was shown to cleave leucine in addition to methionine. Authors in that study have ruled out the PfuMetAP2a activity against leucine substrates and assumed it to be a background reaction contributed by other contaminating proteases. In the current paper, using the pure recombinant enzyme, we report that indeed activity against leucine is directly carried out by the PfuMetAP2a. In addition, the natural product ovalicin which is a specific covalent inhibitor of Type 2 MetAPs does not show efficient inhibition against the PfuMetAP2a. Bioinformatic analysis suggested that a glycine in eukaryotic MetAP2s (G222 in human MetAP2b) and asparagine (N53 in PfuMetAP2a) in archaeal MetAP2s positioned at the analogous position. N53 side chain forms a hydrogen bond with a conserved histidine (H62) at the entrance of the active site and alters its orientation to accommodate the ovalicin. This slight orientational difference of the H62, reduces affinity of the ovalicin by 300,000-fold when compared with the HsMetAP2b inhibition. This difference in the activity is partly reduced in the case of N53G mutation of the PfuMetAP2a.     

Expression and characterization of Mycobacterium tuberculosis methionine aminopeptidase type 1a

Methionine aminopeptidase (MetAP) carries out the cotranslational N-terminal methionine excision and is essential for bacterial survival. Mycobacterium tuberculosis expresses two MetAPs, MtMetAP1a and MtMetAP1c, at different levels in growing and stationary phases, and both are potential targets to develop novel antitubercular therapeutics. Recombinant MtMetAP1a was purified as an apoenzyme, and metal binding and activation were characterized with an activity assay using a fluorogenic substrate. Ni(II), Co(II) and Fe(II) bound tightly at micromolar concentrations, and Ni(II) was the most efficient activator for the MetAP-catalyzed substrate hydrolysis. Although the characteristics of metal binding and activation are similar to MtMetAP1c we characterized before, MtMetAP1a was significantly more active, and more importantly, a set of inhibitors displayed completely different inhibitory profiles on the two mycobacterial MetAPs in both potency and metalloform selectivity. The differences in catalysis and inhibition predicted the significant differences in active site structure.     

Metal-mediated inhibition is a viable approach for inhibiting cellular methionine aminopeptidase

Methionine aminopeptidase (MetAP) plays an essential role for cell survival. Hence, MetAP is a promising target for developing broad spectrum antibacterial agents. MetAP can be activated in vitro by a number of divalent metals, and X-ray structures show that the active site can accommodate two cations. Herein, we demonstrate bacterial growth inhibition by a compound that targets MetAP by recruitment of a third auxiliary metal. Contrary to previous beliefs, this shows that metal-mediated inhibition is a viable approach for discovering MetAP inhibitors that are effective for therapeutic application.     

Discovery of a New Genetic Variant of Methionine Aminopeptidase from Streptococci with Possible Post-Translational Modifications: Biochemical and Structural Characterization

Protein N-terminal methionine excision is an essential co-translational process that occurs in the cytoplasm of all organisms. About 60-70% of the newly synthesized proteins undergo this modification. Enzyme responsible for the removal of initiator methionine is methionine aminopeptidase (MetAP), which is a dinuclear metalloprotease. This protein is conserved through all forms of life from bacteria to human except viruses. MetAP is classified into two isoforms, Type I and II. Removal of the map gene or chemical inhibition is lethal to bacteria and to human cell lines, suggesting that MetAP could be a good drug target. In the present study we describe the discovery of a new genetic variant of the Type I MetAP that is present predominantly in the streptococci bacteria. There are two inserts (insert one: 27 amino acids and insert two: four residues) within the catalytic domain. Possible glycosylation and phosphorylation posttranslational modification sites are identified in the ‘insert one’. Biochemical characterization suggests that this enzyme behaves similar to other MetAPs in terms of substrate specificity. Crystal structure Type Ia MetAP from Streptococcus pneumoniae (SpMetAP1a) revealed that it contains two molecules in the asymmetric unit and well ordered inserts with structural features that corroborate the possible posttranslational modification. Both the new inserts found in the SpMetAP1a structurally align with the P-X-X-P motif found in the M. tuberculosis and human Type I MetAPs as well as the 60 amino acid insert in the human Type II enzyme suggesting possible common function. In addition, one of the β-hairpins within in the catalytic domain undergoes a flip placing a residue which is essential for enzyme activity away from the active site and the β-hairpin loop of this secondary structure in the active site obstructing substrate binding. This is the first example of a MetAP crystallizing in the inactive form.     

Selective inhibition of endothelial cell proliferation by fumagillin is not due to differential expression of methionine aminopeptidases

The angiogenesis inhibitors fumagillin and TNP-470 selectively inhibit the proliferation of endothelial cells, as compared with most other cell types. The mechanism of this selective inhibition remains uncertain, although methionine aminopeptidase-2 (MetAP2) has recently been found to be a target for fumagillin or TNP-470, which inactivates MetAP2 enzyme activity through covalent modification. Primary cultures of human endothelial cells and six other non-endothelial cell types were treated with fumagillin to determine its effect on cell proliferation. Only the growth of endothelial cells was completely inhibited at low concentrations of fumagillin. MetAP1 and MetAP2 levels in these cells were investigated to determine whether differential enzyme expression plays a role in the selective action of fumagillin. Western blot analysis and RT-PCR data showed that MetAP1 and MetAP2 were both expressed in these different types of cells, thus, ruling out differential expression of MetAP1 and MetAP2 as an explanation for the cell specificity of fumagillin. Expression of MetAP2, but not of MetAP1, is regulated. Treatment of human microvascular endothelial cells (HMVEC) with fumagillin resulted in threefold increases of MetAP2 protein in the cells, while MetAP1 remained constant. Similar upregulation of MetAP2 by exposure to fumagillin was also observed in non-endothelial cells, eliminating this response as an explanation for cell specificity. Taken together, these results indicate that while MetAP2 plays a critical role in the effect of fumagillin on endothelial cell proliferation, differential endogenous expression or fumagillin-induced upregulation of methionine aminopeptidases is not responsible for this observed selective inhibition.     

The specificity in vivo of two distinct methionine aminopeptidases in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the essential function of amino-terminal methionine removal is provided cotranslationally by two methionine aminopeptidases (MetAP1 and MetAP2). To examine the individual processing efficiency of each MetAP in vivo, we measured the degree of N-terminal methionine cleavage from a series of mutated glutathione-S-transferase (GST) proteins isolated from yeast wild-type, a map1 deletion strain, a map2 deletion strain, and a map1 deletion strain overexpressing the MAP2 gene. We found that MetAP1 plays the major role in N-terminal methionine removal in yeast. Both MetAPs were less efficient when the second residue was Val, and MetAP2 was less efficient than MetAP1 when the second residue was Gly, Cys, or Thr. These findings indicate that MetAP1 and MetAP2 exhibit different cleavage efficiencies against the same substrates in vivo. Interestingly, although methionine is considered a stabilizing N-terminal residue, we found that retention of the initiator methionine on the Met-Ala-GST mutant protein drastically reduced its half-life in vivo.     

Pyridinylpyrimidines selectively inhibit human methionine aminopeptidase-1

Cellular protein synthesis is initiated with methionine in eukaryotes with few exceptions. Methionine aminopeptidases (MetAPs) which catalyze the process of N-terminal methionine excision are essential for all organisms. In mammals, type 2 MetAP (MetAP2) is known to be important for angiogenesis, while type 1 MetAP (MetAP1) has been shown to play a pivotal role in cell proliferation. Our previous high-throughput screening of a commercial compound library uncovered a novel class of inhibitors for both human MetAP1 (HsMetAP1) and human MetAP2 (HsMetAP2). This class of inhibitors contains a pyridinylpyrimidine core. To understand the structure–activity relationship (SAR) and to search for analogues of 2 with greater potency and higher HsMetAP1-selectivity, a total of 58 analogues were acquired through either commercial source or by in-house synthesis and their inhibitory activities against HsMetAP1 and HsMetAP2 were determined. Through this systematic medicinal chemistry analysis, we have identified (1) 5-chloro-6-methyl-2-pyridin-2-ylpyrimidine as the minimum element for the inhibition of HsMetAP1; (2) 5′-chloro as the favored substituent on the pyridine ring for the enhanced potency against HsMetAP1; and (3) long C4 side chains as the essentials for higher HsMetAP1-selectivity. At the end of our SAR campaign, 25b, 25c, 26d and 30a–30c are among the most selective and potent inhibitors of purified HsMetAP1 reported to date. In addition, we also performed crystallographic analysis of one representative inhibitor (26d) in complex with N-terminally truncated HsMetAP1.     

Yeast glutamine-fructose-6-phosphate aminotransferase (Gfa1) requires methionine aminopeptidase activity for proper function

Methionine aminopeptidase (MetAP) catalyzes the co-translational processing of initiator methionine from nascent proteins. A cellular requirement for MetAP activity is likely due to dysfunction of MetAP substrates that require methionine removal for proper protein function. Glutamine-fructose-6-phosphate aminotransferase (Gfa1) is an essential enzyme in yeast that catalyzes the first and rate-limiting step in hexosamine biosynthesis. The alpha-amino group of Gfa1 Cys-1 has been proposed to act as a nucleophile in the catalytic mechanism. We used two mutational strategies to evaluate whether removal of initiator methionine, catalyzed by MetAP, is required for Gfa1 function. Our results demonstrate that exposure of the alpha-amino group of Cys-1 is required for normal Gfa1 function as failure to do so results in decreased enzyme activity and slow growth. Further, either isoform of MetAP in yeast is sufficient for Gfa1 processing in vivo. These results are the first demonstration of an endogenous yeast protein that requires the exposure of the alpha-amino group by MetAP action for normal function. Additionally, Gfa1 will be a relevant target in therapeutic or physiological applications in which MetAP activity is inhibited.     

Cryo-EM Structures Reveal Relocalization of MetAP in the Presence of Other Protein Biogenesis Factors at the Ribosomal Tunnel Exit

During protein biosynthesis in bacteria, one of the earliest events that a nascent polypeptide chain goes through is the co-translational enzymatic processing. The event includes two enzymatic pathways: deformylation of the N-terminal methionine by the enzyme peptide deformylase (PDF), followed by methionine excision catalyzed by methionine aminopeptidase (MetAP). During the enzymatic processing, the emerging nascent protein likely remains shielded by the ribosome-associated chaperone trigger factor. The ribosome tunnel exit serves as a stage for recruiting proteins involved in maturation processes of the nascent chain. Co-translational processing of nascent chains is a critical step for subsequent folding and functioning of mature proteins.Here, we present cryo-electron microscopy structures of Escherichia coli (E. coli) ribosome in complex with the nascent chain processing proteins. The structures reveal overlapping binding sites for PDF and MetAP when they bind individually at the tunnel exit site, where L22–L32 protein region provides primary anchoring sites for both proteins. In the absence of PDF, trigger factor can access ribosomal tunnel exit when MetAP occupies its primary binding site. Interestingly, however, in the presence of PDF, when MetAP's primary binding site is already engaged, MetAP has a remarkable ability to occupy an alternative binding site adjacent to PDF. Our study, thus, discloses an unexpected mechanism that MetAP adopts for context-specific ribosome association.     

Evidence of a dominant negative mutant of yeast methionine aminopeptidase type 2 in Saccharomyces cerevisiae

Eukaryotic methionine aminopeptidase type 2 (MetAP2, MetAP2 gene (MAP2)), together with eukaryotic MetAP1, cotranslationally hydrolyzes initiator methionine from nascent polypeptides when the side chain of the second residue is small and uncharged. In this report, we took advantage of the yeast (Saccharomyces cerevisiae) map1 null strain's reliance on MetAP2 activity for the growth and viability to provide evidence of the first dominant negative mutant of eukaryotic MetAP2. Replacement of the conserved His(174) with alanine within the C-terminal catalytic domain of yeast MetAP2 eliminated detectable catalytic activity against a peptide substrate in vitro. Overexpression of MetAP2 (H174A) under the strong GPD promoter in a yeast map1 null strain was lethal, whereas overexpression under the weaker GAL1 promoter slightly inhibited map1 null growth. Deletion mutants further revealed that the N-terminal region of MetAP2 (residues 2-57) is essential but not sufficient for MetAP2 (H174A) to fully interfere with map1 null growth. Together, these results indicate that catalytically inactive MetAP2 is a dominant negative mutant that requires its N-terminal region to interfere with wild-type MetAP2 function.     

Metal mediated inhibition of methionine aminopeptidase by quinolinyl sulfonamides

Quinolinyl sulfonamides, such as N-(quinolin-8-yl)methanesulfonamide (10) and N-(5-chloroquinolin-8-yl)methanesulfonamide (11), were identified as potent methionine aminopeptidase (MetAP) inhibitors by high throughput screening of a diverse chemical library of small organic compounds. They showed different inhibitory potencies on Co(II)-, Ni(II)-, Fe(II)-, Mn(II)-, and Zn(II)-forms of Escherichia coli MetAP, and their inhibition is dependent on metal concentration. X-ray structures of E. coli MetAP complexed with 10 revealed that the inhibitor forms a metal complex with the residue H79 at the enzyme active site; the complex is further stabilized by an extended H-bond and metal interaction network. Analysis of the inhibition of MetAP by these inhibitors indicates that this is a typical mechanism of inhibition for many non-peptidic MetAP inhibitors and emphasizes the importance of defining in vitro conditions for identifying and evaluating MetAP inhibitors that will be capable of giving information relevant to the in vivo situation.