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. 2009 Jan;1794(1):137-43.
doi: 10.1016/j.bbapap.2008.09.012. Epub 2008 Oct 7.

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

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

Sanghamitra Mitra et al. Biochim Biophys Acta. 2009 Jan.

Abstract

In order to gain insight into the mechanistic role of a flexible exterior loop near the active site, made up of Y62, H63, G64, and Y65, that has been proposed to play an important role in substrate binding and recognition in the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H63A enzyme was prepared. Mutation of H63 to alanine does not affect the ability of the enzyme to bind divalent metal ions. The specific activity of H63A EcMetAP-I was determined using four different substrates of varying lengths, namely, l-Met-p-NA, MAS, MGMM and MSSHRWDW. For the smallest/shortest substrate (l-Met-p-NA) the specific activity decreased nearly seven fold but as the peptide length increased, the specific activity also increased and became comparable to WT EcMetAP-I. This decrease in specific activity is primarily due to a decrease in the observed k(cat) values, which decreases nearly sixty-fold for l-Met-p-NA while only a four-fold decrease is observed for the tri- and tetra-peptide substrates. Interestingly, no change in k(cat) was observed when the octa-peptide MSSHRWDW was used as a substrate. These data suggest that H63 affects the hydrolysis of small peptide substrates whereas large peptides can overcome the observed loss in binding energy, as predicted from K(m) values, by additional hydrophilic and hydrophobic interactions.

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Figures

Fig. 1
Fig. 1
(A)Space-filling model of EcMetAP-I complexed with the substrate-analog inhibitor (3R)-amino-(2S)-hydroxyheptanoyl- l-Ala- l-Leu- l-Val- l-Phe-OMe (PDB: 3MAT). Color coding: H63A (green, blue and red), S1 pocket residues (magenta), S2 pocket residues (dirty pink) and metal ions (pink). (B) EcMetAP-I active site complexed with the substrate-analog inhibitor (3R)-amino-(2S)-hydroxyheptanoyl- l-Ala- l-Leu- l-Val- l-Phe-OMe (PDB: 3MAT). The H63 residue is 3.66 Å from the third residue of the substrate-analog inhibitor. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2
Fig. 2
Sequence alignment of select MetAP-Is. S1 pocket residues (green) (Ec) Escherichia, coli-I; (St) Salmonella typhimurium-I (all are enterobacteria) (HI) Haemophilus, influenzae-I; (Hp) Helicobacter pylori-I; (Sc) Sacchromyce cerevisiae-I; (Hs) Homo sapiens-I; (Bs) Bacillus subtilis; (Sa) Staphylococcus aureus. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3
Fig. 3
Plot of binding function, r vs. Cs for the binding of Co(II) to H63A EcMetAP-I using MGMM as the substrate in HEPES buffer at pH 7.5. The solid line is a fit to Eq. (3). Inset: plot of specific activity of vs. equivalents of added Co(II) ions.
Fig. 4
Fig. 4
(Top) ITC titration of a 70 μM solution of H63A EcMetAP-I with a 5 mM Co(II) solution. (Bottom) Fit of the ITC data for H63A EcMetAP-I. The fits were obtained using noninteracting sites model and provided an overall n value of 3, with one tight and two loose sites. Reaction conditions: 20 °C in 25 mM HEPES, pH 7.5, and 150 mM KCI.
Fig. 5
Fig. 5
Electronic absorption spectra of a 1 mM H63A EcMetAP-I sample in 25 mM HEPES, pH 7.5, 150 mM KCl in the presence of 0.5 (red), 1.0 (green), 1.5 (blue), and 2 (black) equivalents of Co(II) at 25°C. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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