In the association of serine proteinases with their cognate substrates and inhibitors an important interaction is the fitting of the P₁ side chain of the substrate or inhibitor into a preformed cavity of the enzyme called the S₁ pocket. In turkey ovomucoid third domain, which is a canonical protein proteinase inhibitor, the P₁ residue is Leu¹⁸. Here we report the values of equilibrium constants, K_a, for turkey ovomucoid third domain and 13 additional Leu18X variants with six serine proteinases: bovine α chymotrypsin A, porcine pancreatic elastase, subtilisin Carlsberg, Streptomyces griseus proteinases A and B, and human leukocyte elastase. Eight of the Xs are coded amino acids: Ala, Ser, Val, Met, Gln, Glu, Lys, and Phe, and five are noncoded: Abu, Ape, Ahx, Ahp, and Hse. They were chosen to simplify the interamino acid comparisons. In the homologous series of straight-chain side chains Ala, Abu, Ape, Ahx, Ahp, free energy of binding decreases monotonically with the side-chain length for chymotrypsin with large binding pocket, but even for this enzyme shows curvature. For the two S. griseus enzymes a minimum appears to be reached at Ahp. A minimum is clearly evident for the two elastases, where increasing the side-chain length from Ahx to Ahp greatly weakens binding, but much more so for the apparently more rigid pancreatic enzyme than for the more flexible leukocyte enzyme. β-Branching (Ape/Val) is very deleterious for five of the six enzymes; it is only slightly deleterious for the more flexible human leukocyte elastase. The effect of γ-branching (Ahx/Leu), of introduction of heteroatoms (Abu/Ser), (Ape/Hse), and (Ahx/Met), and of introduction of charge (Gln/Glu) and (Ahp/Lys) are tabulated and discussed. An important component of the free energy of interaction is the distortion of the binding pocket by bulky or branched side chains. Most of the variants studied were obtained by enzymatic semisynthesis. X¹⁸ variants of the 6–18 peptide GlyNH₂ were synthesized and combined with natural reduced peptide 19–56. Disulfide bridges were formed. The GlyNH₂ was removed and the reactive-site peptide bond X¹⁸-Glu¹⁹ was synthesized by complex formation with proteinase K. The resultant complexes were dissociated by sudden pH drop. This kinetically controlled dissociation afforded virgin, reactive-site-intact inhibitor variants.

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