Abstract
Tetracycline (TC) and chlortetracycline (CTC) are common members of the widely used veterinary drug tetracyclines, the residue of which in the environment can enter human body, being potentially harmful. In this study, we establish a new strategy to probe the binding modes of TC and CTC with trypsin based on spectroscopic and computational modeling methods. Both TC and CTC can interact with trypsin with one binding site to form trypsin-TC (CTC) complex, mainly through van der Waals' interactions and hydrogen bonds with the affinity order: TC>CTC. The bound TC (CTC) can result in inhibition of trypsin activity with the inhibition order: CTC>TC. The secondary structure and the microenvironment of the tryptophan residues of trypsin were also changed. However, the effect of CTC on the secondary structure content of trypsin was contrary to that of TC. Both the molecular docking study and the trypsin activity experiment revealed that TC bound into S1 binding pocket, competitively inhibiting the enzyme activity, and CTC was a non-competitive inhibitor which bound to a non-active site of trypsin, different from TC due to the Cl atom on the benzene ring of CTC which hinders CTC entering into the S1 binding pocket. CTC does not hinder the binding of the enzyme substrate, but the CTC-trypsin-substrate ternary complex can not further decompose into the product. The work provides basic data for clarifying the binding mechanisms of TC (CTC) with trypsin and can help to comprehensively understanding of the enzyme toxicity of different members of tetracyclines in vivo.
Highlights
Tetracycline (TC) and chlortetracycline (CTC) are common members of tetracyclines, widely used for disease control and as feed additive in livestock for several decades due to their great therapeutic values [1]
The effect of CTC. The bound TC (CTC) on the secondary structure content of trypsin was contrary to that of TC. Both the molecular docking study and the trypsin activity experiment revealed that TC bound into S1 binding pocket, competitively inhibiting the enzyme activity, and CTC was a non-competitive inhibitor which bound to a non-active site of trypsin, different from TC due to the Cl atom on the benzene ring of CTC which hinders CTC entering into the S1 binding pocket
CTC does not hinder the binding of the enzyme substrate, but the CTC-trypsin-substrate ternary complex can not further decompose into the product
Summary
Tetracycline (TC) and chlortetracycline (CTC) (structure with atom numbers shown in Fig. 1) are common members of tetracyclines, widely used for disease control and as feed additive in livestock for several decades due to their great therapeutic values [1]. The excreted tetracyclines can enter soils, surface and ground water, being a potential risk to human health [3]. The toxicity of tetracyclines residues in the environment including animal food [4], soils [5], and surface and groundwater [3], has attracted widespread attention [6]. The water-soluble globular protein, trypsin (EC 3.4.21.4, structure shown in Figure S1) is a proteolytic enzyme that is excreted by the pancreas into the small intestine and takes part in the digestion of food proteins and other biological processes [7]. Tetracyclines interfere with processes of secretion as well as synthesis of pancreatic protein in pigeons [8], has effect on the secretion kinetics of the rat exocrine pancreas and can lead to the decrease of trypsin level in male wistar rats [9,10]
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