Proton NMR studies of Cucurbita maxima trypsin inhibitors: evidence for pH-dependent conformational change and His25-Tyr27 interaction.
A pH-dependent His25-Tyr27 interaction was demonstrated in the case of Cucurbita maxima trypsin inhibitors (CMTI-I and CMTI-III) by means of nuclear magnetic resonance (NMR) spectroscopy. pH titration, line widths, peak shapes, deuterium exchange kinetics, and two-dimensional nuclear Overhauser effect spectroscopy (NOESY) were employed to characterize a conformational change involving Tyr27, which was shown to be triggered by deprotonation of His25 around pH 6. A hydrogen bond is proposed to be formed between N epsilon of His25 and OH of Tyr27, as a distance between the atoms, His25 N epsilon and Tyr27 OH, of 3.02 A is consistent with a model built with NOE-derived distance constraints. Both the X-ray [Bode, W., Greyling, J.H., Huber, R., Otlewski, J., & Wilusz, T. (1989) FEBS Lett. 242, 282-292] and NMR [Holak, T.A., Gondol, D., Otlewski, J., & Wilusz, T. (1989) J. Mol. Biol. 210, 635-648] structures of CMTI-I at low pH (4.7-5.3) rule out such an interaction between the two aromatic rings, as the ring planes are oriented about 10 A away from each other. The presently characterized relative orientations of His25 and Tyr27 are of functional significance, as these residues make contact with the enzyme in the enzyme-inhibitor complex. Furthermore, trypsin assay and inhibitor-binding studies showed that conformations of trypsin and the squash inhibitor were functionally relevant only in the pH range 6-8. The pKa of His25 was determined and found to be influenced by Glu9/Lys substitution and by the hydrolysis of the reactive-site peptide bond between Arg5 and Ile6.(ABSTRACT TRUNCATED AT 250 WORDS)
Krishnamoorthi
R
R
Department of Biochemistry, Kansas State University, Manhattan 66506.
Lin
C L
CL
Gong
Y X
YX
VanderVelde
D
D
Hahn
K
K
D000595:Amino Acid Sequence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010944:Plants; D011487:Protein Conformation; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000494:pharmacology
D014361:Trypsin Inhibitors; D014357:Trypsin