The small-angle X-ray scattering technique (SAXS) is proposed for the investigation of equilibrium macromolecular interactions of the enzyme-substrate type in solution. Experimental procedures and methods of analysing the data obtained from SAXS have been elaborated. The algorithm for the data analysis allows one to determine the stoichiometric, equilibrium and structural parameters of the enzyme-substrate complexes obtained. The thermodynamic characteristics for the formation of complexes of tRNAphe with phenylalanyl-tRNA synthetase have been determined and demonstrate negative cooperativity for binding of the two tRNAPhe molecules. The structural parameters (I$, &, semi-axes) have been determined for free phenylalanyl-tRNA synthetase and tRNAPhe from E. coli MRE-600 and of enzyme complexes possessing one and two tRNAPhe molecules, indicating structural rearrangements of the enzyme in the interaction with tRNAPhe.Small-angle X-ray scattering; Phenylalanyl-tRNA synthetase; Enzyme-substrate interaction; tRNAPhe

The small‐angle x‐ray scattering (SAXS) technique is used for the investigation of two‐stage equilibrium macromolecular interactions of the enzyme‐substrate type in solution. Experimental procedures and methods of analyzing the data obtained from SAXS have been elaborated. The algorithm for the data analysis allows one to determine the stoichiometric, equilibrium, and structural parameters of the enzyme‐substrate complexes obtained. The thermodynamic characteristics for the formation of complexes of double‐stranded oligonucleotide with Eco dam methyltransferase (MTase) have been determined and demonstrate a high cooperativity of MTase binding when the ternary complex containing the dimeric enzyme is formed. The structural parameters (Rg, Rc, semiaxes) have been determined for free enzyme and polynucleotides and of enzyme‐substrate complexes, indicating structural rearrangements of the enzyme in the interaction with substrates. © 1996 John Wiley & Sons, Inc.