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A 13C-n.m.r. investigation of ionizations within a trypsin-inhibitor complex. Evidence that the pKa of histidine-57 is raised by interaction with the hemiketal oxyanion.

ABSTRACT: The 13C-n.m.r. titration shifts of the alpha-methylene group of N-alkylated imidazoles are shown to be a sensitive probe of the ionization of the imidazolium ion. The 13C-n.m.r. titration shifts of both the intact and denatured/autolysed 2-13C- and 1-13C-enriched trypsin-7-amino-3-benzyloxycarbonylamino-1-chloroheptan-2-one (Z-Lys-CH2Cl) complexes are compared. The titration shift for the denatured/autolysed complex confirms that this ionization is due to deprotonation of the N-alkylated imidazolium ring of histidine-57. In the intact trypsin-inhibitor complex the titration shift due to the 1-13C-enriched carbon is anomalous. We conclude that this titration shift cannot arise solely from the ionization of the imidazolium ion of histidine-57 and that the pKa of the imidazolium ion of histidine-57 is raised in the trypsin-inhibitor complex. The relevance of these studies to the mechanism of action of the serine proteinases is discussed.

SUBMITTER: Primrose WU

PROVIDER: S-EPMC1152802 | biostudies-other | 1985 Nov

REPOSITORIES: biostudies-other

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A 13C-n.m.r. investigation of the ionizations within an inhibitor--alpha-chymotrypsin complex. Evidence that both alpha-chymotrypsin and trypsin stabilize a hemiketal oxyanion by similar mechanisms.

Project description:13C-n.m.r. was used to investigate the structure of the inhibitor enzyme complex formed when alpha-chymotrypsin is alkylated by L-1-chloro-4-phenyl-3-tosylamido-[2-13C]butan-2-one. Two signals are detected. The one at 204.82 +/- 0.11 p.p.m. does not titrate from pH 3 to 9 and is assigned to alkylated methionine-192. The second signal titrates from 99.08 p.p.m. to 103.44 p.p.m. with pKa 8.67. This signal is assigned to a tetrahedral adduct formed between the hydroxy group of serine-195 and the inhibitor. The titration shift of the tetrahedral adduct is ascribed to the ionization of the hemiketal hydroxy group. It is proposed that the resulting oxyanion is stabilized by interaction with the imidazolium ion of histidine-57. It is argued that this interaction must raise the pKa of at least 70% of histidine-57 to greater than 11. On denaturation/autolysis of the inhibitor-enzyme complex neither of the signals associated with the intact complex is detected, but a new signal is observed that titrates from 203.52 p.p.m. to 206.08 p.p.m. with pKa = 5.27. This titration shift is assigned to the ionization of the imidazolium ion of alkylated histidine, confirming that the inhibitor has alkylated histidine-57. The significance of these results for the catalytic mechanism of the serine proteinases is discussed.

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Trypsin

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Catalytic activity of -chymotrypsin in which histidine-57 has been methylated.

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The pKa of the catalytic histidine residue of chloramphenicol acetyltransferase.

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pKa Determination of a Histidine Residue in a Short Peptide Using Raman Spectroscopy.

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Propionate metabolism in the rat heart by 13C n.m.r. spectroscopy.

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