¹³C NMR Study of How the Oxyanion pK_a Values of Subtilisin and Chymotrypsin Tetrahedral Adducts Are Affected by Different Amino Acid Residues Binding in Enzyme Subsites S₁−S₄^,

Abstract

A range of substrate-derived chloromethane inhibitors have been synthesized which have one to four amino acid residues. These have been used to inhibit both subtilisin and chymotrypsin. Using 13C NMR, we have shown that all except one of these inhibitors forms a tetrahedral adduct with chymotrypsin, subtilisin, and trypsin. From the pH-dependent changes in the chemical shift of the hemiketal carbon of the tetrahedral adduct, we are able to determine the oxyanion pKa in the different inhibitor derivatives. Our results suggest that in both the subtilisin and chymotrypsin chloromethane derivatives the oxyanion pKa is largely determined by the type of amino acid residue occupying the S1, subsite while binding in the S2-S4 subsites only has minor effects on oxyanion pKa values. Using free energy relationships, we determine that the different R groups of the amino acid residues binding in the S1 subsite only have minor effects on the oxyanion pKa values. We propose that the lower polarity of the chymotrypsin active site relative to that of the subtilisin active site explains why the oxyanion pKa is higher and more sensitive to the type of chloromethane inhibitor used in the chymotrypsin derivatives than in the subtilisin derivatives.

Oxyanion and tetrahedral intermediate stabilisation by subtilisin: Detection of a new tetrahedral adduct

“…The active site of subtilisin can be described as a shallow groove open on one side to solvent while that of chymotrypsin consists of a more deeply invaginated hydrophobic pocket [16,31,32]. This could explain why the glyoxal aldehyde carbon is dehydrated in chymotrypsin inhibitor complexes but it is partially hydrated in the more solvent exposed subtilisin-glyoxal inhibitor complexes.…”

“…was observed. It has been shown that in chymotrypsin-or subtilisin-chloromethylketone inhibitor tetrahedral adducts the oxyanion pK a values are ~2 pK a units smaller in the subtilisinchloromethylketone adducts compared to chymotrypsin adducts [11,16,33]. Therefore it has been argued [22] that the hemiketal oxyanion in species (e) in Scheme 1 in the subtilisin-BPN'-Z-Ala-Pro-Phe-glyoxal complex will have a pK a of ~ 2.5 at least 2 pK a units lower than that observed (pK a ~ 4.5) in chymotrypsin -glyoxal inhibitor complexes [20,21].…”

“…Using 13 C-NMR it has been shown that when substrate derived chloromethylketone inhibitors alkylate the active site histidine residue of the serine proteases trypsin [12,13], chymotrypsin [10,11,[14][15][16] and subtilisin [10,11,16] the active site serine hydroxy group reacts with ketone carbon to form a tetrahedral adduct analogous to the catalytic tetrahedral intermediate.…”

“…Oxyanion pK a values were 2 pK a units lower in subtilisin chlomethylketone adducts compared to chymotrypsin chloromethylketone inhibitor adducts suggesting that oxyanion stabilisation is more effective in subtilisin compared to chymotrypsin [16]. However, X-ray crystallographic studies have shown that the alkylation of the active site histidine by the chloromethylketone inhibitor causes significant changes in the structure of the tetrahedral adduct compared to tetrahedral adducts which do not alkylate the active site histidine [17,18].…”

Substrate Recognition

“…Earlier crystallographic studies [84,85] had suggested a weak hydrogen bond involving Asn155 in the Michaelis complex that becomes stronger in the transition state for acylation. The existence of the oxyanion hole in chymotrypsin (Gly193 and Ser195) proposed by Henderson [86] received support from crystal structures of combinations with transition state analogs [87] and from 13 C-NMR studies by the Malthouse group (e.g., [88][89][90][91]) on the enzymes derivatized by substrate-derived inhibitors. It is of particular interest that the oxyanion pK a values of chymotrypsin and subtilisin in the tetrahedral adducts depend on binding interactions in the recognition sites of these enzymes [90].…”