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How a Fragment Draws Attention to Selectivity Discriminating Features between the Related Proteases Trypsin and
Anna Sandner1, Khang Ngo1, Johannes Schiebel1
1Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, 35032 Marburg, Germany.
Journal of Medicinal Chemistry
|January 20, 2021
Summary
Serine proteases thrombin and trypsin show subtle S1 pocket differences, including a sodium ion in thrombin, influencing substrate selectivity. These structural and electrostatic variations, not single factors, dictate their distinct enzymatic activities.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Serine proteases thrombin and trypsin share structural similarities in their S1 pockets, including Asp189.
- Despite similarities, they exhibit distinct substrate specificities: thrombin cleaves after Arg, while trypsin cleaves after Lys and Arg.
Purpose of the Study:
- To investigate the structural and electrostatic factors governing the substrate selectivity of thrombin and trypsin.
- To elucidate the role of specific amino acid substitutions (Ala190Ser, Glu192Gln) and the thrombin-specific sodium ion in S1 pocket function.
Main Methods:
- Crystallography was employed to determine the structures of enzymes with various ligands.
- Isothermal Titration Calorimetry (ITC) was used to measure binding affinities.
- Mutated enzyme variants were studied to assess the impact of specific residues.
Main Results:
- The S1 pockets of thrombin and trypsin possess conserved replacements (Ala190Ser, Glu192Gln) with no observed affinity changes.
- Thrombin features a unique Na+-binding site adjacent to Asp189, absent in trypsin.
- Benzylamine forms hydrogen bonds with Asp189 in trypsin and Glu192 in thrombin.
- An electrostatic gradient created by Glu192 and the sodium ion in thrombin's S1 pocket is crucial for selectivity.
Conclusions:
- Enzyme selectivity is determined by a combination of factors, including amino acid composition, electrostatic potential, and solvation patterns within the S1 pocket.
- The thrombin-specific sodium ion and Glu192 play a significant role in establishing an electrostatic gradient that contributes to thrombin's substrate specificity.
- Subtle differences in the S1 pocket environment, rather than a single dominant feature, dictate the distinct cleavage preferences of thrombin and trypsin.
