Complex of .alpha.-chymotrypsin and N-acetyl-L-leucyl-L-phenylalanyl trifluoromethyl ketone: structural studies with NMR spectroscopy

Abstract

A dipeptidyl trifluoromethyl ketone, N-acetyl-L-leucyl-L-[1-13C]phenylalanyl trifluoromethyl ketone, was synthesized. This compound inhibits chymotrypsin with Ki = 1.2 microM [Imperiali B., & Abeles, R.H. (1986) Biochemistry 25, 3760-3767]. The complex formed between this inhibitor and alpha-chymotrypsin was examined with 1H, 13C, and 19F NMR spectroscopy to establish its structure in solution. The keto group of the trifluoro ketone is present as an ionized hemiketal group as deduced from the comparison of its 13C chemical shift with those of model hemiketals. The pKa of the hemiketal hydroxyl in the complex is approximately 4.9, which is about 4.2 units lower than the pKa of model hemiketals. This observation provides direct evidence that serine proteases are able to stabilize the oxyanions of tetrahedral adducts. Evidence is also presented for the presence of an Asp-His H bond and protonation of the imidazole group of His-57 in the tetrahedral adduct. The pKa of His-57 is higher than 10. This observation directly indicates that the pKa of His-57 is elevated in a complex containing a tetrahedral adduct.

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

“…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].…”

High-Precision Measurement of Hydrogen Bond Lengths in Proteins by Nuclear Magnetic Resonance Methods

“…SSHBs have been proposed as participants in the mechanisms of action of hydrolase enzymes, lyases, a ligase, and several isomerases. 16 NMR spectroscopic evidence for strong hydrogen bonding has been obtained in serine proteases, [17][18][19][20][21][22][23][24] aspartate amino transferase, 25,26 ketosteroid isomerase, 27,28 and triosephosphate isomerase 29 ( Fig. 2).…”

Enzyme–substrate interaction in the catalytic triad of serine proteases: increase in the pKa of Asp102

“…Further, the significance of Asp"!# has been shown by site-directed mutagenesis [33][34][35]. Studies with some tetrahedral-intermediate models show an increase in the pK a of His&( during the catalytic process [8,16,[28][29][30], and it has been suggested that, in addition to the hydrogen bonding in the ' oxyanion hole ', the zwitterionic tetrahedral intermediate is stabilized by favourable interaction of the oxyanion with the imidazolium ion of His&( and, further, that the pK a of His&( is raised because of electrostatic\hydrogen-bond interaction with Asp"!# after substrate binding [15,16].…”

“…Liang and Abeles [30] detected an increase in the pK a of His&( ( 10) in the complex formed between α-chymotrypsin and Nacetyl--leucyl--phenylalanyl trifluoromethyl ketone. Accordingly, an increase in the pK a of His&( in a real tetrahedral adduct was also suggested.…”

“…In the following discussion I will show experimental evidence for the change in the pK a of Asp"!# by reinterpreting previous NMR [5,6] and isotope-effect [26,27] studies. pK a adjustment has been previously proposed for ionizing groups of serine proteases [8,16,[28][29][30][31], but direct experimental evidence for a change in the pK a of Asp"!# has not been described. Asp"!# has been suggested to ensure the correct tautomeric form by hydrogen bonding and the optimal orientation of the imidazole ring toward serine hydroxy group and, further, to allow electrostatic stabilization of the imidazolium ion formed during the reaction [1,5,31].…”