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...

Comparative CD and Fourier transform ir (FTIR) spectroscopic data on N-Boc protected linear peptides with or without the (Pro-Gly) beta-turn motif (e.g., Boc-Tyr-Pro-Gly-Phe-Leu-OH and Boc-Tyr-Gly-Pro-Phe-Leu-OH) are reported herein. The CD spectra, reflecting both backbone and aromatic contributions, were not found to be characteristic of the presence of beta-turns. In the amide I region of the FTIR spectra, analyzed by self-deconvolution and curve-fitting methods, the beta-turn band showed up between 1639 and 1633 cm-1 in trifluoroethanol (TFE) but only for models containing the (Pro-Gly) core. This band was also present in the spectra in chloroform but absent in dimethylsulfoxide. These findings, in agreement with recent ir data on cyclic models and 3(10)-helical polypeptides and proteins in D2O [see S. J. Prestrelski, D. M. Byler, and M. P. Thompson (1991), International Journal of Peptide and Protein Research, Vol. 37, pp. 508-512; H. H. Mantsch, A. Perczel, M. Hollósi, and G. D. Fasman (1992), FASEB Journal, Vol. 6, p. A341; H. H. Mantsch, A. Perczel, M. Hollósi, and G. Fasman (1992), Biopolymers, Vol. 33, pp. 201-207; S. M. Miick, G. V. Martinez, W. R. Fiori, A. P. Todd, and G. L. Millhauser (1992), Nature, Vol. 359, pp. 653-655], suggest that the amide I band, with a major contribution from the acceptor C = O of the 1<--4 intramolecular H bond of beta-turns, appears near or below 1640 cm-1, rather than above 1660 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)

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A kappa-opioid receptor subtype was purified from a digitonin extract of frog brain membranes, using affinity chromatography. The affinity resin was prepared by coupling dynorphin (1-10) to AH Sepharose 4B. The purified receptor binds 4,750 pmol [3H]ethylketocyclazocine (EKC) per mg protein (5,600-fold purification over the membrane-bound receptor) with a Kd of 9.1 nM. The addition of cholesterol-phosphatidylethanolamine (2:1) enhanced 3.6-fold the binding activity of the purified material, which gives a purification very close to the theoretical. The purified receptor protein exhibits high affinity for kappa-selective ligands. The purified fraction shows one major band (65,000 Mr) in sodium dodecyl sulfate (SDS) gel electrophoresis.