Trypsin (Tr) and chymotrypsin (Ch) have similar tertiary structures, yet Tr cleaves peptides at arginine and lysine residues and Ch prefers large hydrophobic residues. Although replacement of the S1 binding site of Tr with the analogous residues of Ch is sufficient to transfer Ch specificity for ester hydrolysis, specificity for amide hydrolysis is not transferred. Trypsin is converted to a Ch-like protease when the binding pocket alterations are further modified by exchange of the Ch surface loops 185 through 188 and 221 through 225 for the analogous Tr loops. These loops are not structural components of either the S1 binding site or the extended substrate binding sites. This mutant enzyme is equivalent to Ch in its catalytic rate, but its substrate binding is impaired. Like Ch, this mutant utilizes extended substrate binding to accelerate catalysis, and substrate discrimination occurs during the acylation step rather than in substrate binding.

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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 binding of human alpha1-proteinase inhibitor to rat trypsin was shown by NMR spectroscopy to raise the pKa' of His57 in the active site but not to disrupt the hydrogen bond between His57 and Asp102. Similar NMR results were observed for the Asp189 to serine mutant of rat trypsin, which is much more stable than wild-type trypsin against autoproteolysis as the result of mutation of the residue at the base of the specificity pocket. This mutant was used in further studies aimed at determining the extent of the conformational transition in trypsin that accompanies serpin binding and leads to disruption of the catalytic activity of the proteinase such that the inhibitor complex is trapped at the acyl enzyme intermediate stage. The stability of rat trypsin toward thermal denaturation was found to be lower in the free enzyme than in the complex with alpha1-proteinase inhibitor. This suggests that the complex contains extensive protein-protein interactions that stabilize overall folding. On the other hand, previous investigations have shown that the proteinase in serpin-proteinase complexes becomes more susceptible to limited proteolysis, suggesting that the conformational change that accompanies binding leads to the exposure of susceptible loops in the enzyme. The existence of this type of conformational change upon complex formation has been confirmed here by investigation of the rate of cleavage of disulfide linkages by added dithiothreitol. This study revealed that, despite the increased stability of trypsin in the complex, one or more of its disulfide bridges becomes much more easily reduced. We suggest that the process of complex formation with alpha1-proteinase inhibitor converts trypsin D189S into an inactive, loose structure, which serves as a "conformational trap" of the enzyme that prevents catalytic deacylation. It is also proposed that plastic region(s) of the activation domain of trypsin may play a crucial role in this inhibitor-induced structural rearrangement.

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D-Gluco- and D-xylopyranosylidene-spiro-hydantoins and -thiohydantoins were prepared from the parent sugars in a six-step, highly chemo-, regio-, and stereoselective procedure. In the key step of the syntheses C-(1-bromo-1-deoxy-beta-D-glycopyranosyl)formamides were reacted with cyanate ion to give spiro-hydantoins with a retained configuration at the anomeric center as the major products. On the other hand, thiocyanate ions gave spiro-thiohydantoins with an inverted anomeric carbon as the only products. On the basis of radical inhibition studies, a mechanistic rationale was proposed to explain this unique stereoselectivity and the formation of C-(1-hydroxy-beta-D-glycopyranosyl)formamides as byproducts. Enzyme assays with a and b forms of muscle and liver glycogen phosphorylases showed spiro-hydantoin 12 and spiro-thiohydantoin 14 to be the best and equipotent inhibitors with K(i) values in the low micromolar range. The study of epimeric pairs of D-gluco and D-xylo configurated spiro-hydantoins and N-(D-glucopyranosyl)amides corroborated the role of specific hydrogen bridges in binding the inhibitors to the enzyme.

A 2 B, and A2B block copolymers, where A ) polyisobutylene, B ) poly-(methyl vinyl ether), and the superscripts denote molecular weight asymmetry, with constant molecular weight and composition have been synthesized by living cationic polymerization. The influence of architecture on aqueous micellar properties of these block copolymers were investigated in the temperature range 20-30 °C by fluorescence spectroscopy and static and dynamic light scattering (SLS and DLS). The critical micelle concentration (cmc) measured at 23 °C increased in the order A 2B < A 1 A 2 B < AB. The partition equilibrium constants, Kv of pyrene, characteristic of hydrophobicity, increased in the opposite order of cmc. The hydrodynamic radii (Rh) and aggregation numbers (Nagg) of micelles remained approximately constant in the whole temperature range for A 1 A 2 B and A2B and below 25 °C for AB. At ∼25 °C, however, there was a sudden increase in both Rh and Nagg for AB. Below 25 °C both Rh and Nagg increased in the order AB < A 1 A 2 B < A2B. The particle size distribution for all block copolymers remained narrow in the whole temperature range. The results are discussed in terms of possible morphologies.