15N and 1H NMR Spectroscopy of the Catalytic Histidine in Chloromethyl Ketone-Inhibited Complexes of Serine Proteases

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Abstract
The hemiketal hydroxyl groups in chloromethyl ketone (cmk) complexes of trypsin and chymotrypsin have been reported to ionize to the oxyanion with pKa values 2−4 pKa units below expectations for such a functional group on the basis of the behavior of the hemiketal carbon atom in 13C NMR spectra [Finucane, M. D., & Malthouse, J. P. G. (1992) Biochem. J. 286, 889−900]. The low pKa indicates the enzymes selectively stabilize the oxyanion form of the bound inhibitor, and therefore that cmk complexes may be good models of enzyme-mediated transition-state stabilization. However, the 13C NMR studies could not rule out His57 as the titrating group. Here we report the behavior of the ring 15N atoms of His57 in the Ala-Ala-Pro-Val−cmk complex of α-lytic protease. Both Nδ1 and Nε2 of His57 respond to an ionization with a pKa of ∼7.5, but His57 itself does not titrate as Nε2 remains alkylated and Nδ1 remains bonded to a proton over the entire pH range. The species titrating with a pKa of ∼7.5 must therefore be the hemiketal hydroxyl. The results also show that the 1H NMR signal from the proton in the Asp-His hydrogen bond behaves in a characteristic manner in cmk complexes and can be used diagnostically to confirm that His57 does not titrate and to measure the pKa of the hemiketal hydroxyl in cmk−protease complexes without resorting to 15N-labeling. We have used the behavior of this signal to directly confirm that His57 does not titrate in the trypsin and chymotrypsin complexes that were the subjects of the original 13C NMR studies.

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