SECONDARY STRUCTURE OF RECOMBINANT HUMAN CYSTATHIONINE BETA-SYNTHASE IN AQUEOUS-SOLUTION - EFFECT OF LIGAND-BINDING AND PROTEOLYTIC TRUNCATION

The secondary structural composition and substrate-induced conformatio nal changes of recombinant human cystathionine beta-synthase (CBS) in aqueous solution have been investigated in its full-length form (tetra mer of 63-kDa subunits) by Fourier transform infrared (FT-IR) and circ ular dichroism (CD) spectroscopies. In addition, structural comparison of a proteolytic truncated form (dimer of 45-kDa subunits) to that of the full-length enzyme has also been carried out, Second-derivative a nd Fourier self-deconvolutional enhanced infrared spectra revealed ami de I band components ascribed to beta-sheet (1689, 1638, and 1627 cm(- 1)), alpha-helix (1658 cm(-1)), beta-turn (1679 and 1668 cm(-1)), and unordered (1658 cm(-1)) structures in the spectra of the full-length e nzyme. Quantitative analysis of FT-IR and CD spectra reveals that the full-length enzyme consists of about 48-53% beta-sheet, 25-30% alpha-h elix, 8-10% turn, and 10-19% unordered structures, Under constraint of the spectroscopic data, theoretical prediction of locations of these secondary structural elements using Garnier's method shows that human CBS may contain a beta-sheet/alpha-helix/beta-sheet core structure, Se cond-derivative spectrum of the truncated enzyme exhibited all the maj or spectral features that are present in the full-length enzyme, indic ating a preservation of the core structure of the enzyme. Significant differences were observed between the infrared spectra of the enzymes with or without the substrate, serine, indicating a substrate-induced conformational change in the enzyme, which did not result in a change in over all composition of secondary structural content based on quant itative analysis of FT-IR and far-UV CD spectre. (C) 1997 Academic Pre ss.