APPLICATION OF THE SMALL-ANGLE X-RAY-SCATTERING TECHNIQUE FOR THE STUDY OF 2-STEP EQUILIBRIUM ENZYME-SUBSTRATE INTERACTIONS

The small-angle x-ray, scattering (SAXS) technique is used for the inv estigation of two-stage equilibrium macromolecular interactions of the enzyme-substrate type in solution. Experimental procedures and method s of analyzing the data obtained from SAXS have been elaborated The al gorithm for the data analysis allows one to determine the stoichiometr ic, equilibrium, and structural parameters of the enzyme-substrate com plexes obtained. The thermodynamic characteristics for the formation o f complexes of double-stranded oligonucleotide with Eco dam methyltran sferase (MTase) have been determined and demonstrate a high cooperativ ity of MTase binding when the ternary complex containing the dimeric e nzyme is formed. The structural parameters (R(g), R(c), semiaxes) have been determined for free enzyme and polynucleotides and of enzyme-sub strate complexes, indicating structural rearrangements of the enzyme i n the interaction with substrates. (C) 1996 John Wiley & Sons, Inc.