Electrostatic Complementarity within the Substrate-Binding Pocket of Trypsin

• Published in: Proceedings of the National Academy of Sciences - PNAS 1988-07, Vol.85 (14), p.4961-4965
• Main Authors: Gráf, László, Jancsó, Ágnes, Szilágyi, László, Hegyi, György, Pintér, Katalin, Náray-Szabó, Gábor, Hepp, József, Medzihradszky, Kálmán, Rutter, William J.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Analytical, structural and metabolic biochemistry; Animals; Aspartic Acid; Binding Sites; Biochemistry; Biological and medical sciences; Catalysis; Computer Graphics; Electrochemistry; Electrostatics; Enzyme substrates; Enzymes; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Gibbs free energy; Hydrolases; Hydrolysis; Kinetics; Lysine; Molecular Sequence Data; Mutation; Rats; Serine; Structure-Activity Relationship; Substrate Specificity; Thermodynamics; Trypsin - genetics; Trypsin - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.85.14.4961

The aspartic residue (Asp-189) at the base of the substrate-binding pocket of trypsin was replaced by serine (present in a similar position in chymotrypsin) through site-directed mutagenesis. The wild-type (with Asp-189 in the mature trypsin sequence) and mutant (Ser-189) trypsinogens were expressed in Escherichia coli, purified to homogeneity, activated by enterokinase, and tested with a series of fluorogenic tetrapeptide substrates with the general formula succinyl-Ala-Ala-Pro-Xaa-AMC, where AMC is 7-amino-4-methylcoumarin and Xaa is Lys, Arg, Tyr, Phe, Leu, or Trp. As compared to [Asp189]trypsin, the activity of [Ser189]trypsin on lysyl and arginyl substrates decreased by about 5 orders of magnitude while its Km values increased only 2- to 6-fold. In contrast, [Ser189]trypsin was 10-50 times more active on the less preferred, chymotrypsin-type substrates (tyrosyl, phenylalanyl, leucyl, and tryptophanyl). The activity of [Ser189]trypsin on lysyl substrate was about 100-fold greater at pH 10.5 than at pH 7.0, indicating that the unprotonated lysine is preferred. Assuming the reaction mechanisms of the wild-type and mutant enzymes to be the same, we calculated the changes in the transition-state energies for various enzyme-substrate pairs to reflect electrostatic and hydrogen-bond interactions. The relative binding energies (E) in the transition state are as follows: EII > EPP > EPA > EIP≈ EIA, where I = ionic, P = nonionic but polar, and A = apolar residues in the binding pocket. These side-chain interactions become prominent during the transition of the Michaelis complex to the tetrahedral transition-state complex.