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13C-NMR study of the inhibition of delta-chymotrypsin by a tripeptide-glyoxal inhibitor.

ABSTRACT: A new inhibitor, Z-Ala-Pro-Phe-glyoxal (where Z is benzyloxycarbonyl),has been synthesized and shown to be a competitive inhibitor of delta-chymotrypsin, with a K(i) of 25+/-8 nM at pH 7.0 and 25 degrees C. Z-Ala-Pro-[1-(13)C]Phe-glyoxal and Z-Ala-Pro-[2-(13)C]Phe-glyoxal have been synthesized, and (13)C-NMR has been used to determine how they interact with delta-chymotrypsin. Using Z-Ala-Pro-[2-(13)C]Phe-glyoxal we have detected a signal at 100.7 p.p.m. which we assign to the tetrahedral adduct formed between the hydroxy group of Ser-195 and the (13)C-enriched keto-carbon of the inhibitor. This signal is in a pH-dependent slow exchange with a signal at 107.6 p.p.m. which depends on a pK(a) of approximately 4.5, which we assign to oxyanion formation. Thus we are the first to detect an oxyanion pK(a) in a reversible chymotrypsin-inhibitor complex. A smaller titration shift of 100.7 p.p.m. to 103.9 p.p.m. with a pK(a) of approximately 5.3 is also detected due to a rapid exchange process. This pK(a) is also detected with the Z-Ala-Pro-[1-(13)C]Phe-glyoxal inhibitor and gives a larger titration shift of 91.4 p.p.m. to 97.3 p.p.m., which we assign to the ionization of the hydrated aldehyde hydroxy groups of the enzyme-bound inhibitor. Protonation of the oxyanion in the oxyanion hole decreases the binding efficiency of the inhibitor. From this decrease in binding efficiency we estimate that oxyanion binding in the oxyanion hole reduces the oxyanion pK(a) by 1.3 pK(a) units. We calculate that the pK(a)s of the oxyanions of the hemiketal and hydrated aldehyde moieties of the glyoxal inhibitor are both lowered by 6.4-6.9 pK(a) units on binding to chymotrypsin. Therefore we conclude that oxyanion binding in the oxyanion hole has only a minor role in decreasing the oxyanion pK(a). We also investigate how the inhibitor breaks down at alkaline pH, and how it breaks down at neutral pH in the presence of chymotrypsin.

SUBMITTER: Djurdjevic-Pahl A

PROVIDER: S-EPMC1222393 | biostudies-other | 2002 Mar

REPOSITORIES: biostudies-other

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Project description:L-1-Chloro-4-phenyl-3-tosylamido[1-13C]butan-2-one (Tos-[1-13C]Phe-CH2Cl) and Tos-[1-13C,2H2]Phe-CH2Cl were prepared and used to alkylate delta-chymotrypsin. The relaxation parameters of the 13C-n.m.r. signal resulting from the alkylation of histidine-57 in both enzyme-inhibitor complexes were determined at 1.88 T and 6.34 T as well as the spin-lattice relaxation times of the backbone alpha-carbon atoms of the unenriched Tos-Phe-CH2-delta-chymotrypsin complex. It is concluded that the species examined do not have significant internal librational motions and that the rotational correlation time of the monomeric enzyme-inhibitor complex is 16.0 +/- 3.2 ns. The signal from the 13C-enriched atom of Tos-[1-13C,2H2]Phe-CH2Cl is split into a quintet (JCD = 23 Hz) whereas in the Tos-[1-13C,2H2]Phe-CH2-delta-chymotrypsin complex the signal from the 13C-enriched inhibitor carbon atom is decoupled. This decoupled signal had linewidths of 16 +/- 3 Hz and 52 +/- 2 Hz at 1.88 T and 6.34 T respectively, whereas linewidths at 40 +/- 2 Hz and 53 +/- 4 Hz were obtained for the same signal in the Tos-[1-13C]Phe-CH2-delta-chymotrypsin complex at 1.88 T and 6.34 T respectively. Therefore whereas deuteration produces a 2.5-fold reduction in linewidth at 1.88 T there is no significant decrease in the linewidth at 6.34 T. This result is explained by using the rigid rotor model, which predicts that the quadrupolar spin-lattice relaxation rate will be faster at low field strengths, resulting in more efficient deuterium decoupling by scalar relaxation of the second kind at lower field strengths. It is also predicted that deuterium decoupling by scalar relaxation will become less efficient as rotational correlation times increase. The consequences of these predictions for the detection of 13C-enriched atomic probes of proteins are discussed. It is also shown that a spin-echo pulse sequence can be used to remove signals due to protonated carbon atoms without attenuating the signal due to deuterated carbon atoms.

Project description:delta-Chymotrypsin has been alkylated by 1-13C- and 2-13C-enriched tosylphenylalanylchloromethane. In the intact inhibitor derivative, signals due to the 1-13C- and 2-13C-enriched carbon atoms have chemical shifts which titrate from 55.10 to 59.50 p.p.m. and from 99.10 to 103.66 p.p.m. respectively with similar pKa values of 8.99 and 8.85 respectively. These signals are assigned to a tetrahedral adduct formed between the hydroxy group of serine-195 and the inhibitor. An additional signal at 58.09 p.p.m. and at 204.85 p.p.m. in the 1-13C- and 2-13C-enzyme-inhibitor derivatives respectively does not titrate when the pH is changed and it is assigned to alkylated methionine-192. On denaturation/autolysis of the 1-13C-enriched enzyme-inhibitor derivative these signals associated with the intact inhibitor derivative are no longer detected, and a new signal, which titrates from 56.28 to 54.84 p.p.m. with a pKa of 5.26, is detected. The titration shift of this signal is assigned to the deprotonation of the imidazolium cation of alkylated histidine-57 in the denatured/autolysed enzyme-inhibitor derivative. Model compounds which form stable hydrates and hemiketals in aqueous solutions have been synthesized. By comparing the 13C titration shifts of these model compounds with those of the 13C enriched trypsin- and delta-chymotrypsin-inhibitor derivatives, we deduce that, in both of the intact enzyme-inhibitor derivatives, the zwitterionic tetrahedral adduct containing the imidazolium cation of histidine-57 and the hemiketal oxyanion predominates at alkaline pH values. It is estimated that in both the trypsin and delta-chymotrypsin-inhibitor derivatives the concentration of this zwitterionic tetrahedral adduct is 10,000-fold greater than it would be in water. We conclude that the pKa of the oxyanion of the hemiketal in the presence of the imidazolium cation of histidine-57 is 7.9 and 8.9 in the trypsin and delta-chymotrypsin-inhibitor derivatives respectively and that the pKa of the imidazolium cation of histidine-57 is greater than 7.9 and greater than 8.9 when the oxyanion is present as its conjugate acid, whereas, when the oxyanion is present, the pKa of the imidazolium cation is greater than 11 in both enzyme-inhibitor derivatives. We discuss how these enzymes preferentially stabilize zwitterionic tetrahedral adducts in the intact enzyme-inhibitor derivatives and how they could stabilize similar tetrahedral intermediates during catalysis. It is suggested that substrate binding could raise the pKa of the imidazolium cation of histidine-57 before tetrahedral-intermediate formation which would explain the enhanced nucleophilicity of the hydroxy group of serine-195.(ABSTRACT TRUNCATED AT 400 WORDS)

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13C-NMR study of the inhibition of delta-chymotrypsin by a tripeptide-glyoxal inhibitor.

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