Electrostatic complementarity within the substrate-binding pocket of trypsin.

Abstract

The aspartic residue at the base of the substrate-binding pocket of trypsin was replaced by serine (present in a similar position in chymotrypsin) through sitedirected mutagenesis. The wild-type (with 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 succinylAla-Ala-Pro-Xaa-AMC, where AMC is 7-amino-4-methylcoumarin and Xaa is Lys, Arg, Tyr, Phe, Leu, or Trp. As compared to [Asp'l8trypsin, the activity of [Ser'"1trypsin on lysyl and arginyl substrates decreased by about 5 orders of magnitude while its Km values increased only 2-to 6-fold. In contrast, [Serl89]trypsin was 10-50 times more active on the less preferred, chymotrypsin-type substrates (tyrosyl, phenylalanyl, leucyl, and tryptophanyl). The activity of [Ser"]9]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: El, > 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.The binding of substrates or inhibitors to the specificity pocket of an enzyme involves a combination of chemical forces including hydrogen bonds and electrostatic, hydrophobic, and steric interactions. The complexity of the interactions involved in the substrate specificity of an enzyme is exemplified by trypsin. The three-dimensional structures of trypsin bound to pancreatic trypsin inhibitor (PTI) (1)(2)(3)(4) or to the pseudosubstrate benzamidine (5, 6) suggest that the carboxylate of Asp-189, at the base of the trypsin binding pocket, is largely responsible for the specificity of binding of the enzyme to positively charged amino acid side chains.The major role of electrostatic interactions in the trypsin binding pocket has been analyzed by measuring (7) and calculating (8) the stabilization energies of binding between a series of benzamidine analogs and trypsin. In addition, the high degree of structural similarity of the trypsin and chymotrypsin binding pockets (9, 10) is consistent with the experimental observations that aromatic side chains may form favorable hydrophobic interactions with the trypsin binding pocket (10-13 by using a series of synthetic fluorogenic substrates with various amino acids in the C-terminal (P1) position in order to compare the electrostatic interactions of the different enzyme-substrate pairs. MATERIALS AND METHODSMaterials. Tetrapeptide substrates with the fluorogenic leaving...

Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin

“…Serine proteases of tryptic specificity vary substantially in their preferences for Arg versus Lys (78,79), and it is common for a large part of the substrate discrimination of a serine protease to be implemented through k cat rather than K m (66). However, mesotrypsin appears unique among trypsins in the degree of preference for Arg over Lys at the P 1 position because discrimination between these residues is minimal for both rat anionic trypsin (80,81) and bovine trypsin (78); in this regard, mesotrypsin resembles more highly selective serine proteases, such as tissue plasminogen activator (79,82).…”

Amino acid sequence determination of Ancrod, the thrombin‐like α‐fibrinogenase from the venom of Akistrodon rhodostoma

“…Expression systems are available for both prokaryotic and ¢ukaryotic sources, The number of sulfhydryls in Ancrod suggest that renaturation from E. coil inclusion bodies will be challenging. Although trypsin has been successfully expressed in an active form in the periplasmic space [16], significant quantities of such sulfhydryl-rich proteins may orily be available from eukaryotic cell systems.…”

Attempts to convert chymotrypsin to trypsin

“…SBTI-Sepharose, MUTMAC, MUGB, AMC and 7-methylumbelliferon were from Sigma Chemical Co. The tetrapeptide substrates with AMC fluorogenic leaving group were prepared as described [9].…”

“…Suprisingly enough, the corresponding mutations in trypsin did not transform the specificity: although the trypsin activity measured on polypeptide amide substrates were already completely gone on an Aspl89Ser substitution [9], no significant chymotrypsin-like activity was observed. Moreover, the tryptic activity of this mutant returned when non-covalently bound acetate ion was present in the pocket [lo].…”