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

Abstract

An active site aspartate residue, Asp97, in the methionine aminopeptidase (MetAPs) from Escherichia coli (EcMetAP-I) was mutated to alanine, glutamate, and asparagine. Asp97 is the lone carboxylate residue bound to the crystallographically determined second metal-binding site in EcMetAP-I. These mutant EcMetAP-I enzymes have been kinetically and spectroscopically characterized. Inductively coupled plasma-atomic emission spectroscopy analysis revealed that 1.0 +/- 0.1 equivalents of cobalt were associated with each of the Asp97-mutated EcMetAP-Is. The effect on activity after altering Asp97 to alanine, glutamate or asparagine is, in general, due to a approximately 9000-fold decrease in k(ca) towards Met-Gly-Met-Met as compared to the wild-type enzyme. The Co(II) d-d spectra for wild-type, D97E and D97A EcMetAP-I exhibited very little difference in form, in each case, between the monocobalt(II) and dicobalt(II) EcMetAP-I, and only a doubling of intensity was observed upon addition of a second Co(II) ion. In contrast, the electronic absorption spectra of [Co_(D97N EcMetAP-I)] and [CoCo(D97N EcMetAP-I)] were distinct, as were the EPR spectra. On the basis of the observed molar absorptivities, the Co(II) ions binding to the D97E, D97A and D97N EcMetAP-I active sites are pentacoordinate. 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. Although mononuclear MetAPs are active, the physiologically relevant form of the enzyme is probably dinuclear, given that the majority of the data reported to date are consistent with weak cooperative binding.

Fig. 1

Active site of EcMetAP-I showing the metal-binding residues, including Asp97. Prepared from Protein Data Bank file 2MAT.

Fig. 2

Amino acid sequence alignment for selected MetAPs, prolidase and aminopeptidase P (AMPP). Prepared from Protein Data Bank files 1C21, 1QXZ, 1O0X, 1XGO, 1BN5, 1PV9, and 1A16.

Fig. 3

ITC titration of 70 µm solution of D97E EcMetAP-I with a 5 mm Co(II) solution at 25 °C in 25 mm Hepes (pH 7.5) and 150 mm KCl.

Fig. 4

Electronic absorption spectra of 1 mm wild-type (black), D97A (green), D97N (blue) and D97E (red) EcMetAP-I with increments of one and two equivalents of Co(II) in 25 mm Hepes buffer (pH 7.5) and 150 mm KCl.

Fig. 5

Binding function r versus C_S, the concentration of free metal ions in solution for D97E EcMetAP-I in 25 mm Hepes buffer (pH 7.5) and 150 mm KCl at three different wavelengths. The solid lines correspond to fits of each data set to Eqn (3).

Fig. 6

Co(II)-EPR of EcMetAP-I and variants. Traces A, B and D are the EPR spectra of [CoCo(WT-EcMetAP-I)] (A), [Co_(D97E-EcMetAP-I)] (B), and [CoCo(D97E-EcMetAP-I)] (D). Trace C is a computer simulation of B assuming two species. The major species exhibited resolved hyperfine coupling and was simulated with spin Hamiltonian parameters S = 3/ 2, M_S = |± 1/ 2〉, g_x,y = 2.57, g_z = 2.67, D >> g BS (50 cm-1), E / D = 0.185, A_y = 9.0 × 10⁻³ cm⁻¹. The minor species was best simulated (g_x,y = 2.18, g_z = 2.6, E / D = 1/ 3, A_y (unresolved) = 4.5 × 10⁻³ cm⁻¹) assuming some unresolved hyperfine coupling, although no direct evidence for this was obtained. Trace E is of spectrum D with arbitrary amounts of spectrum A subtracted. Trace F is the experimental EPR spectrum of [CoCo(D97E- EcMetAP-I)] recorded in parallel mode (B₀║ B₁). Traces G and I are spectra of [Co_(D97N- EcMetAP-I)], and traces H and J are spectra of [CoCo(D97N- EcMetAP-I)]; the insert of H shows the hyperfine region of G expanded. Trace K is the spectrum of [Co_(D97A- EcMetAP-I)], and L is of [CoCo(D97A-EcMetAP-I)]. Spectra A, B, D and I–K were recorded using 0.2 mW power at 8 K. Spectrum F was recorded using 20 mW at 8 K, and spectra G and H were recorded using 2 mW at 6 K. Trace G is shown × 2 compared to H, I is shown × 2 compared to J, and K is shown × 2 compared to L. Other intensities are arbitrary. Spectrum F was recorded at 9.37 GHz whereas all other experimental spectra were at 9.64 GHz.