An additivity-based sequence to reactivity algorithm for the interaction of members of the Kazal family of protein inhibitors with six selected serine proteinases is described. Ten consensus variable contact positions in the inhibitor were identified, and the 19 possible variants at each of these positions were expressed. The free energies of interaction of these variants and the wild type were measured. For an additive system, this data set allows for the calculation of all possible sequences, subject to some restrictions. The algorithm was extensively tested. It is exceptionally fast so that all possible sequences can be predicted. The strongest, the most specific possible, and the least specific inhibitors were designed, and an evolutionary problem was solved.

In the association of serine proteinases with their cognate substrates and inhibitors an important interaction is the fitting of the PI side chain of the substrate or inhibitor into a preformed cavity of the enzyme called the SI pocket. In turkey ovomucoid third domain, which is a canonical protein proteinase inhibitor, the PI residue is Leu18. Here we report the values of equilibrium constants, K,, for turkey ovomucoid third domain and 13 additional Leul8X variants with six serine proteinases: bovine 01 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. 0-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 y-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. X" variants of the 6-18 peptide GlyNH, were synthesized and combined with natural reduced peptide 19-56. Disulfide bridges were formed. The GlyNH, was removed and the reactive-site peptide bond Xls-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.Keywords: binding of amino acid side chains; deformability of binding pockets; enzymatic semisynthesis; noncoded amino acid residues; ovomucoid third domains; serine proteinases Abbreviations: P I , the residue contributing the carbonyl portion to the reactive-site peptide bond; SI, the pocket in the enzyme to which the P I residue binds; OMXXX3, ovomucoid third domain, with XXX designating the species; TKY, turkey; SVP, silver pheasant; SWN, black swan; WTD, West Indian tree duck; MNQ, Montezuma quail; X"OMTKY3, X residue replaces Leu" in OMTKY3 (Fig. 1); Abu CY, aminobutyric acid; Ape CY, aminopentanoic acid (norvaline); Ahx CY, aminohexanoic acid (norleucine); Ahp a , aminoheptanoic acid; H...