Evidence that proline focuses movement of the floppy loop of arylalkylamine N-acetyltransferase (EC 2.3.1.87)

Abstract

Arylalkylamine N-acetyltransferase (AANAT) catalyzes the N-acetylation of serotonin, the penultimate step in the synthesis of melatonin. Pineal AANAT activity increases at night in all vertebrates, resulting in increased melatonin production. This increases circulating levels of melatonin, thereby providing a hormonal signal of darkness. Kinetic and structural analysis of AANAT has determined that one element is floppy. This element, termed Loop 1, is one of three loops that comprise the arylalkylamine binding pocket. During the course of chordate evolution, Loop 1 acquired the tripeptide CPL, and the enzyme became highly active. Here we focused on the functional importance of the CPL tripeptide and found that activity was markedly reduced when it was absent. Moreover, increasing the local flexibility of this tripeptide region by P64G and P64A mutations had the counterintuitive effect of reducing activity and reducing the overall movement of Loop 1, as estimated from Langevin dynamics simulations. Binding studies indicate that these mutations increased the off-rate constant of a model substrate without altering the dissociation constant. The structural kink and local rigidity imposed by Pro-64 may enhance activity by favoring configurations of Loop 1 that facilitate catalysis and do not become immobilized by intramolecular interactions.

FIGURE 1.

The root mean square deviation of superimposed C_α atoms of residues Thr-41—Cys-77, which include Loop 1. Results of dynamics simulated for the wild-type, P64A mutant, and P64G mutant of ovine AANAT are shown. All deviations were calculated relative to the AANAT crystal structure (PDB code 1B6B). rmsd, root mean square deviation.

FIGURE 2.

Schema of protein structure. A, position of CoA-T inhibitor (blue) in the active center of AANAT (14). The structure of Loop 1 is highlighted in red, and amino acids mutated during this study are marked in yellow and labeled with symbols. B and C show differences between the organization and position of Loop 1 without (B) and with inhibitor (C). Pictures are rotated several degrees compared with the panel A. Loop 1 is in red, and Pro-64 is highlighted in yellow.

FIGURE 3.

Denaturation of proteins in guanidine HCl. Fluorescence of 0.3 μm protein samples in increasing concentrations of guanidine HCl. The buffer used contained 0.1 m ammonium acetate, pH 6.8, 25 mm NaCl, and 2 mm TCEP. The exposure of tryptophans in the structure was monitored by fluorescence (excitation = 285 nm, emission = 339 nm). The similar shape of the denaturation curves indicates the similar and homogenous folding among all prepared protein samples. Curves are representative of several experiments.

FIGURE 4.

Enzyme activity as a function of tryptamine concentration. The enzyme activity of the indicated proteins was measured as a function of the tryptamine concentration. The enzyme assay contained 0.5 mm [³H]Ac-CoA and was performed as described under “Experimental Procedures.” K_m and V_max values appear in Table 1.

FIGURE 5.

Characterization of bisubstrate inhibitors. A, the fluorescence excitation spectra of 2.8 μm CoA-T and CoA-HNE were determined (excitation wavelength from 250 to 360 nm, emission wavelength = 420 nm). The spectrum of CoA-HNE has a peak between 305 and 350 nm not seen with CoA-T (and also not seen in protein), allowing the former to be used as a fluorescence probe in binding experiments. The buffer used was 0.1 m ammonium acetate, pH 6.8, containing 25 mm NaCl and 2 mm TCEP. Emission is given as arbitrary units. B, inhibition of acetylation activity of ovine wild-type AANAT using the indicated concentrations of CoA-T and CoA-HNE. The assay contained GST-ovine AANAT-(2–207) (4 nm), 10 mm tryptamine, and 0.5 mm [³H]Ac-CoA. Activity was measured as described under “Experimental Procedures.” CoA-T and CoA-HNE had similar inhibitory potency.

FIGURE 6.

Binding of CoA-HNE to studied proteins. Fluorescence anisotropy as a function of protein concentration added to 3 μm solution of CoA-HNE is shown. The buffer used was 0.1 m ammonium acetate, pH 6.8, containing 25 mm NaCl and 2 mm TCEP. Curves characterize the binding of the probe to the indicated proteins. Derived K_d values are in Table 1.

FIGURE 7.

Displacement of inhibitors. Fluorescence anisotropy is shown as a function of time. Samples of proteins (2 μm) were 75–85% saturated with CoA-HNE. After 5 min the fluorescence anisotropy was measured. At t = 200 s, CoA-T was added to a final concentration of 170 μm, and fluorescence anisotropy measurement was resumed. The buffer was 0.1 m ammonium acetate, pH 6.8, containing 25 mm NaCl and 2 mm TCEP. The k_off values calculated from the rate of change of fluorescence anisotropy are presented in Table 1.