Conformation of a synthetic hexapeptide substrate of collagen lysyl hydroxylase
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The hexapeptide Hyp-Gly-Pro-Lys-Gly-Glu was synthesized as a potential substrate for collagen lysyl hydroxylase. Kinetic data on the interaction of this peptide with purified chicken embryo lysyl hydroxylase showed that the hexapeptide is a moderately good substrate having Km, Vmax, and Kcat/Km values comparable to those of synthetic peptide substrates having longer chain lengths. Circular dichroism spectral data suggested a consecutive beta turn or 3(10) helical conformation for the peptide in trifluoroethanol. The two-dimensional 1H-TOCSY spectrum of the peptide in dimethylsulfoxide permitted complete assignment of all the protons in the hexapeptide. Through-space connectivities between protons in the peptide molecule were obtained from two-dimensional 1H-NOESY spectral data on the peptide. Using the distances calculated from these data as input constraints, the minimum-energy conformation of the peptide was computed. These calculations and an unconstrained Monte Carlo molecular simulation both led to a folded conformation for the hexapeptide with dihedral angles close to a set of consecutive beta turns as the lowest-energy conformer. This structure is stabilized further by a salt bridge between the side chains of Lys4 and Glu6. Several other conformers energetically close to the minimum-energy conformer exhibited the structural features of the latter except for variations at the N-terminal end and in the side chains. In conjunction with data obtained earlier on lysyl hydroxylase (P. Jiang and V. S. Ananthanarayanan, 1991, J. Biol. Chem. 266, 22960-22967) and the functionally related prolyl hydroxylase (P. L. Atreya and V. S. Ananthanarayanan, 1991, J. Biol. Chem. 266, 2852-2858), the present results suggest that the folded beta turn in the respective peptide substrate may be the structural determinant at the catalytic sites of these enzymes. Additional structural features may govern the effective binding of the peptide at the enzymes' active sites.
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