Effects of acrylamide on the activity and structure of human brain creatine kinase

Abstract

Acrylamide is widely used worldwide in industry and it can also be produced by the cooking and processing of foods. It is harmful to human beings, and human brain CK (HBCK) has been proposed to be one of the important targets of acrylamide. In this research, we studied the effects of acrylamide on HBCK activity, structure and the potential binding sites. Compared to CKs from rabbit, HBCK was fully inactivated at several-fold lower concentrations of acrylamide, and exhibited distinct properties upon acrylamide-induced inactivation and structural changes. The binding sites of acrylamide were located at the cleft between the N- and C-terminal domains of CK, and Glu232 was one of the key binding residues. The effects of acrylamide on CK were proposed to be isoenzyme- and species-specific, and the underlying molecular mechanisms were discussed.

Keywords: acrylamide; docking simulation; human brain creatine kinase; inactivation.

Figure 1.

Structure of HBCK monomer (PDB ID: 3B6R) [20]. N and C denote the N- and C-terminus of the protein. The positions of the substrates are highlighted by the stick model.

Figure 2.

Effect of acrylamide on the activity of HBCK. The residual activity was measured after 2 h incubation of HBCK in 50 mM Tris-HCl buffer, pH 8.0, with the addition of various concentrations of acrylamide at 25 °C. The final enzyme concentration was 2 μM. The data are presented as average ± standard errors for three repetitions.

Figure 3.

Inactivation kinetics of HBCK by various concentrations of acrylamide ranging from 0 to 800 mM. The enzyme solutions were mixed with various concentrations of acrylamide, and aliquots were taken at the indicated time points. Then the residual activity was measured using the standard activity assay, and the data were normalized by taking the activity recorded at 0 min as 100%. The data were fitted by a biphasic process, and the fitted data are presented as solid lines. The rate constants are presented in Table 1.

Figure 4.

Effect of acrylamide on the ANS fluorescence of HBCK. The enzyme solutions were mixed with various concentrations of acrylamide and equilibrated for 2 h. The final concentration of ANS was 40 μM, and the solutions were incubated at ambient temperature for 30 min in the dark before measurements. The final enzyme concentration was 2 μM. The presented spectra were obtained by subtracting the spectra of ANS in the same buffer.

Figure 5.

Native-PAGE analysis of the tertiary structural changes of HBCK induced by acrylamide. The protein was dissolved in 50 mM Tris-HCl buffer, pH 8.0, in the presence of various concentrations of acrylamide. Lanes 1–6 indicate the protein incubated in the buffer with the addition of 0, 50, 100, 200, 400 and 800 mM acrylamide, respectively.

Figure 6.

The surface of HBCK (A and B) and acrylamide binding sites predicted by Autodock (C and D) and Fred (E and F). (A and B) The surface of the HBCK molecule. The yellow parts indicated the position of the cleft or pocket between the two domains of HBCK. Panel (B) shows the top view of the structure shown in panel (A). (C-F) The residues forming the binding site are shown by a line model, the acrylamide molecule is presented by a space-filling model, while Glu232 is highlighted by a stick model.