13C and 1H NMR Studies of Ionizations and Hydrogen Bonding in Chymotrypsin-Glyoxal Inhibitor Complexes

Abstract

Benzyloxycarbonyl (Z)-Ala-Pro-Phe-glyoxal and Z-Ala-AlaPhe-glyoxal have both been shown to be inhibitors of ␣-chymotrypsin with minimal K i values of 19 and 344 nM, respectively, at neutral pH. These K i values increased at low and high pH with pK a values of ϳ4.0 and ϳ10.5, respectively. By using surface plasmon resonance, we show that the apparent association rate constant for Z-Ala-Pro-Phe-glyoxal is much lower than the value expected for a diffusion-controlled reaction.13 C NMR has been used to show that at low pH the glyoxal keto carbon is sp 3 -hybridized with a chemical shift of ϳ100.7 ppm and that the aldehyde carbon is hydrated with a chemical shift of ϳ91.6 ppm. The signal at ϳ100.7 ppm is assigned to the hemiketal formed between the hydroxy group of serine 195 and the keto carbon of the glyoxal. In a slow exchange process controlled by a pK a of ϳ4.5, the aldehyde carbon dehydrates to give a signal at ϳ205.5 ppm and the hemiketal forms an oxyanion at ϳ107.0 ppm. At higher pH, the re-hydration of the glyoxal aldehyde carbon leads to the signal at 107 ppm being replaced by a signal at 104 ppm (pK a ϳ9.2). On binding either Z-Ala-Pro-Phe-glyoxal or Z-AlaAla-Phe-glyoxal to ␣-chymotrypsin at 4 and 25°C, 1 H NMR is used to show that the binding of these glyoxal inhibitors raises the pK a value of the imidazolium ion of histidine 57 to a value of >11 at both 4 and 25°C. We discuss the mechanistic significance of these results, and we propose that it is ligand binding that raises the pK a value of the imidazolium ring of histidine 57 allowing it to enhance the nucleophilicity of the hydroxy group of the active site serine 195 and lower the pK a value of the oxyanion forming a zwitterionic tetrahedral intermediate during catalysis.Specific substrate-derived glyoxal inhibitors have been shown to be potent inhibitors of the serine proteinases (1-4). Z 4 -Ala-Pro-Phe-glyoxal is an extremely potent reversible inhibitor of ␦-chymotrypsin with an apparent disassociation constant of 25 Ϯ 8 nM at pH 7.0 (1).The ␣-keto carbon of the glyoxal inhibitor is expected to occupy the same position as the carbonyl carbon of a substrate, and it has been shown that it is bound as a tetrahedral adduct, which should closely resemble the tetrahedral intermediate formed during substrate catalysis (1). By using 13 C NMR, it has been shown that ␦-chymotrypsin (1) and subtilisin (2) reduce the oxyanion pK a by ϳ6 and ϳ8 pK a units, respectively. It has been estimated that hydrogen bonding in the oxyanion hole will only reduce the oxyanion pK a by ϳ1.3 pK a units (1). This is consistent with the fact that hydrogen bonding is expected to be effective in both water and in the oxyanion hole, and so it should not reduce the oxyanion pK a to a value lower than that expected in water. This has led to the conclusion that hydrogen bonding in the oxyanion hole only has a minor role in lowering the oxyanion pK a (5-7). However, it has been proposed that substrate binding raises the pK a of the imidazolium ion of the active site histidine enabling ...

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

“…The K i values for the inhibition of subtilisin Carlsberg by Z-Ala-Ala-Pheglyoxal increased at higher and lower pHs (Table 1). Similar increases in K i values were observed when chymotrypsin was inhibited by either Z-Ala-Pro-Phe-glyoxal [20,21] or ZAla-Ala-Phe-glyoxal [21].…”

“…3B) as has been observed with chymotrypsin-glyoxal inhibitor complexes [21]. But, with chymotrypsin-glyoxal inhibitor complexes the decrease in intensity on decreasing the pH led to the concomitant increase in the intensity of a signal that titrated from ~100 to ~104 p.p.m.…”

“…But, with chymotrypsin-glyoxal inhibitor complexes the decrease in intensity on decreasing the pH led to the concomitant increase in the intensity of a signal that titrated from ~100 to ~104 p.p.m. [20,21]. However, as in our earlier study with the Z-Ala-Pro-Phe-glyoxal and subtilisin the signals at 100 to 104 p.p.m.…”

“…(Fig. 3B) [21]) observed for the hemiketal ionisation by slow exchange in the Z-Ala-Pro-Phe-glyoxal-chymotrypsin complex.…”

“…to one at 107.6 p.p.m. and it depends on a pK a of ~4.5 [20,21]. With subtilisin and Z-Ala-Pro-Phe-glyoxal a signal at ~107 p.p.m.…”