Thermodynamic Criterion for the Conformation of P₁ Residues of Substrates and of Inhibitors in Complexes with Serine Proteinases

Abstract

Eglin c, turkey ovomucoid third domain, and bovine pancreatic trypsin inhibitor (Kunitz) are all standard mechanism, canonical protein inhibitors of serine proteinases. Each of the three belongs to a different inhibitor family. Therefore, all three have the same canonical conformation in their combining loops but differ in their scaffoldings. Eglin c (Leu45 at P1) binds to chymotrypsin much better than its Ala45 variant (the difference in standard free energy changes on binding is -5.00 kcal/mol). Similarly, turkey ovomucoid third domain (Leu18 at P1) binds to chymotrypsin much better than its Ala18 variant (the difference in standard free energy changes on binding is -4.70 kcal/mol). As these two differences are within the +/-400 cal/mol bandwidth (expected from the experimental error), one can conclude that the system is additive. On the basis that isoenergetic is isostructural, we expect that within both the P1 Ala pair and the P1 Leu pair, the conformation of the inhibitor's P1 side chain and of the enzyme's specificity pocket will be identical. This is confirmed, within the experimental error, by the available X-ray structures of complexes of bovine chymotrypsin Aalpha with eglin c () and with turkey ovomucoid third domain (). A comparison can also be made between the structures of P1 (Lys+)15 of bovine pancreatic trypsin inhibitor (Kunitz) ( and ) and of the P1 (Lys+)18 variant of turkey ovomucoid third domain (), both interacting with chymotrypsin. In this case, the conformation of the side chains is strikingly different. Bovine pancreatic trypsin inhibitor with (Lys+)15 at P1 binds to chymotrypsin more strongly than its Ala15 variant (the difference in standard free energy changes on binding is -1.90 kcal/mol). In contrast, turkey ovomucoid third domain variant with (Lys+)18 at P1 binds to chymotrypsin less strongly than its Ala18 variant (the difference in standard free energies of association is 0.95 kcal/mol). In this case, P1 Lys+ is neither isostructural nor isoenergetic. Thus, a thermodynamic criterion for whether the conformation of a P1 side chain in the complex matches that of an already determined one is at hand. Such a criterion may be useful in reducing the number of required X-ray crystallographic structure determinations. More importantly, the criterion can be applied to situations where direct determination of the structure is extremely difficult. Here, we apply it to determine the conformation of the Lys+ side chain in the transition state complex of a substrate with chymotrypsin. On the basis of kcat/KM measurements, the difference in free energies of activation for Suc-AAPX-pna when X is Lys+ and X is Ala is 1.29 kcal/mol. This is in good agreement with the corresponding difference for turkey ovomucoid third domain variants but in sharp contrast to the bovine pancreatic trypsin inhibitor (Kunitz) data. Therefore, we expect that in the transition state complex of this substrate with chymotrypsin, the P1 Lys+ side chain is deeply inserted into the enzyme's specificity pocket as it is in th...

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

“…However, for nonadditivity of P 1 mutations versus scaffold or PЈ 2 mutations, our data are less easily explained and contrast with previous studies. Prior investigations of the association of canonical inhibitors mutated at the P 1 position with various serine proteases have generally shown interscaffolding additivity or similar energetic effects of a P 1 mutation irrespective of scaffolding context (67,69,86). The primary exception occurs in rare cases where different scaffolds impose alternative binding conformations for the P 1 residue (69,86), a situation that we do not expect here.…”

“…Prior investigations of the association of canonical inhibitors mutated at the P 1 position with various serine proteases have generally shown interscaffolding additivity or similar energetic effects of a P 1 mutation irrespective of scaffolding context (67,69,86). The primary exception occurs in rare cases where different scaffolds impose alternative binding conformations for the P 1 residue (69,86), a situation that we do not expect here. Because our calculated ⌬G I values are all fairly small, one possible explanation for the apparent nonadditivity could be underestimation of the margin of error in our K i determinations.…”

Characterization of potato proteinase inhibitor II reactive site mutants

“…As appears from the improved affinity of AAA-ARA relative to AAA-AAA, the P1 arginine contributes considerably to the affinity for chymotrypsin. This would suggest that the P1 arginine of PI-2 is not protonated under our experimental conditions (pH 7.8), and that it is energetically favorable to embed its side chain in the apolar S1 pocket of chymotrypsin, like in the case of Lys18± OMTKY3 [40].…”

Molecular Characterization ofAncylostoma ceylanicumKunitz-Type Serine Protease Inhibitor: Evidence for a Role in Hookworm-Associated Growth Delay

“…Tight-binding inhibitors act via a mechanism that involves interaction of the enzyme's reactive site with the P1 amino acid residue of the inhibitor (43,44,51). Although this interaction is reversible, the enzyme-inhibitor complex is sufficiently stable that considerable inhibition can be detected at nearly equimolar concentrations.…”

“…Based on an alignment of AceKI with other members of the Kunitz-type family, we identified Met 26 as the likely P1 inhibitory reactive site amino acid (8,43,49,66). Therefore, in order to more clearly elucidate the role of Met 26 in defining the spectrum of inhibition of AceKI, a series of site-directed mutants were produced that had various substitutions incorporated at the P1 site (23,33,41,46,51,65). These recombinant proteins were expressed and purified, and their inhibitory activities against the three serine protease targets of AceKI were measured.…”