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...

The distribution and properties of frog brain kappa-opioid receptor subtypes differ not only from those of the guinea pig brain, but also from that of the rat brain. In guinea pig cerebellum the kappa 1 is the dominant receptor subtype, frog brain contains mainly the kappa 2 subtype, and the distribution of the rat brain subtypes is intermediate between the two others. In competition experiments it has been established that ethylketocyclazocine and N-cyclopropylmethyl-norazidomorphine, which are nonselective kappa-ligands, have relatively high affinities to frog brain membranes. The kappa 2 ligands (Met5)enkephalin-Arg6-Phe7 and etorphine also show high affinities to the frog brain. Kappa 1 binding sites measured in the presence of 5 microM/D-Ala2-Leu5/enkephalin represent 25-30% of [3H]ethylketocyclazocine binding in frog brain membranes. The kappa 2 subtype in frog brain resembles more to the mu subtype than the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and /D-Ala2-(Me)Phe4-Gly5-ol/enkephalin (DAGO). From our data it is evident that the opioid receptor subtypes are already present in the amphibian brain but the differences among them are less pronounced than in mammalian brain.

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.

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