Proton NMR studies of Cucurbita maxima trypsin inhibitors: Evidence for pH-dependent conformational change and his25 - try27 interaction

Journal Article · Tue Jan 28 00:00:00 EST 1992 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00118a037· OSTI ID:5488858

Krishnamoorthi, R; Lin, Chanlan Sun; Gong, Yuxi ^[1]; VanderVelde, D ^[2]; Hahn, K ^[3]

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1. Kansas State Univ., Manhattan (United States)
2. Univ. of Kansas, Lawrence (United States)
3. Univ. of Colorado, Denver (United States)

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{sub {epsilon}} of His25 and OH of Tyr27, as a distance between the atoms, His25 N{epsilon} and Tyr25 OH, of 3.02 {angstrom} is consistent with a model built with NOE-derived distance constraints. 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 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 pK{sub a} 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. As these sites are located far (>10 {angstrom}) from His25, the results point out conformational changes that are propagated to a distant site in the protein molecule.

• OSTI ID: 5488858
• Journal Information: Biochemistry; (United States), Vol. 31:3; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ENZYME INHIBITORS
NUCLEAR MAGNETIC RESONANCE
HISTIDINE
CONFORMATIONAL CHANGES
TYROSINE
ENZYME ACTIVITY
OVERHAUSER EFFECT
PROTONS
TRYPSIN
AMINO ACIDS
AZOLES
BARYONS
CARBOXYLIC ACIDS
ELEMENTARY PARTICLES
ENZYMES
FERMIONS
HADRONS
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HYDROLASES
HYDROXY ACIDS
IMIDAZOLES
MAGNETIC RESONANCE
NUCLEONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PEPTIDE HYDROLASES
PROTEINS
RESONANCE
SERINE PROTEINASES
550201* - Biochemistry- Tracer Techniques