The effect of N-terminal acetylation and the inhibition activity of acetylated enkephalins on the aminopeptidase M-catalyzed hydrolysis of enkephalins

Abstract

High performance liquid chromatography and high performance liquid chromatography/electrospray ionization-mass spectrometry were used to study the effect of N-terminal acetylation and the inhibition activity of acetylated enkephalins on the aminopeptidase M (EC 3.4.11.2)-catalyzed hydrolysis of methionine (Met-enk) and leucine enkephalins (Leu-enk). Acetylation imparts a significant enhancement in the proteolytic stability of these two peptides. After 30 min of the reaction, < 10% of both acetylated enkephalins was hydrolyzed. In an 8-h incubation period, only a maximum of 54% acetylated (Ac)-Met-enk and 38% Ac-Leu-enk was hydrolyzed. Vmax and Km [infil] for the degradation of Ac-Met-enk were 1.4 nmol/min/50 ng and 2.2 mM, respectively. The corresponding values for the reaction of Ac-Leu-enk were 0.5 nmol/min/50 ng and 0.9 mM. Also, the aminopeptidase M activity on Met-enk can be inhibited in the presence of Ac-Met-enk, which acts as a mixed-type inhibitor with the inhibition constant (K(i)) of I x 10(-3) M.

Fig. 1

Various enzymes involved in the degradation of enkephalins.

Fig. 2

HPLC chromatograms showing separation of the products of the reaction of aminopeptidase M with Ac-Met-enk (detection at 280 nm). The products were separated isocratically by using 20% acetonitrile in 10 mM ammonium acetate at a flow rate of 1.5 ml/min. (a) Incubation for a period of 10 min—no reaction products are seen, only Ac-Met-enk elutes at 3.9 min. (b) Incubation period of 1 h—only 20% of the substrate is hydrolyzed. The peaks eluting at 2.0 and 3.9 min are due to Ac-Tyr and Ac-Met-enk, respectively.

Fig. 3

The rate of disappearance of Ac-Met-enk (circles) and appearance of Ac-Tyr (squares). Ac-Met-enk was reacted with aminopeptidase M at 37°C and pH 7.02. % Peak area, the % peak area of Ac-Tyr or Ac-Met-enk relative to the sum of the peak areas of Ac-Tyr and Ac-Met-enk.

Fig. 4

The rate of disappearance of Ac-Leu-enk (circles) and appearance of Ac-Tyr (squares). Ac-Leu-enk was reacted with aminopeptidase M at 37°C and pH 7.02. %Peak area, the % peak area of Ac-Tyr or Ac-Met-enk relative to the sum of the peak areas of Ac-Tyr and Ac-Leu-enk.

Fig. 5

The total ion current chromatograms obtained by on-line HPLC/ESI/MS. The reaction products were separated isocratically by using 12% acetonitrile in ammonium acetate at a flow rate of 1 ml/min. Only 200 μl/min of the solvent was allowed to enter the ESI source. (a) The hydrolysis of Met-enk-only Tyr, FM, and GGFM were detected at retention times of 4.0, 5.0, and 6.1 min, respectively. Met-enk and YGG were not detected. (b) The hydrolysis of Met-enk in the presence of Ac-Met-enk- molecular ions of Tyr, GGFM, Met-enk, and, Ac-Met-enk detected at 4.0, 6.1, 9.2, and 11.3 min, respectively. Ac-Tyr, YGG, and FM were not detected.

Fig. 6

Comparison of the Ac-Met-enk-inhibited reaction (filled squares) with the noninhibited reaction (filled circles). V, reaction rate in nmol/min/50 ng; [S], [Met-enk] in mM. (a) Plot of the reaction rate vs. [Met-enk]. (b) The Hanes–Woolf plots for the inhibited and noninhibited reactions.

Fig. 7

The Lineweaver–Burk plots for the inhibited reaction (dotted line) and the noninhibited reaction (dark line). These plots were drawn based upon the K_{m [infi]} and V_max values obtained from the Hanes–Woolf plots in Fig. 6b. V and [S] are the same as in Fig. 6.