Proton magnetic resonance study of Streptomyces subtilisin inhibitor. pH titration and assignments of individual tyrosyl resonances

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Abstract
The pH titration at 25.degree. C and the assignment of aromatic proton resonances of 3 tyrosyl residues of each identical subunit of Streptomyces subtilisin inhibitor (Mr [molecular ratio] 23,000; isolated from S. albogriseolus S-3523) by high-resolution PMR spectroscopy is reported. The complete assignments of the specific tyrosyl resonances were made based on the results of the differential chemical modification of the tyrosyl residues with tetranitromethane followed by peptide analysis, independently of the knowledge of the crystal structure. The pKa values of Tyr-7, -75, and -93 in a 2H2O solution were 10.95, 11.8 and .gtorsim. 12.6, respectively, at 25.degree. C, whereas pKa values of nitrated Tyr-7 and -75 were 7.3 and 7.9, respectively. Tyr-93 was not nitratable under normal conditions. The strong resistance to nitration, together with the extremely high pKa value and the high-field shifted positions of the ring proton resonances of Tyr-93 at neutral pH, strongly suggests that Tyr-93 takes part in an H bonding as a proton donor. Tyr-7 is more easily modified with tetranitromethane than Tyr-75, although in the crystal Tyr-75 is more exposed than Tyr-7. The result, together with the pKa value of Try-75 significantly higher than that of a normal tyrosine, indicates that the microenvironment of Tyr-75 is more restricted in solution than in solid. Structural details of a protein may be quite similar in solution to those in the crystal in the rigid hydrophobic region of the protein, but in the surface region of the protein, local structures may well differ between the solution and the crystal.