Time-Resolved Crystal Structure Analysis of Glutathione Synthetase

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One of the major goals of structural biology should perhaps be to achieve 4-dimensional structure determination (with time being the 4th dimension).In order to apply time resolved crystallography to glutathione synthetase from E.coli, we determined a crystal structure of the enzyme by X-ray diffraction method. The enzyme is a tetramer with four identical Subunits of 316 amino acid residues. The loop from Ile-226 to Arg-241 in the enzyme is rich in glycine and alanine and too flexible to take a fixed conformation. The apparent function of the loop is to serve as a lid that shields the reaction intermediate, gamma-glutamylcysteinylphosphate, from the solvent water.Kinetic diffraction studies require the fast measurement of a sequence of 3-dimensional sets of structure factors with an accuracy and completeness that is sufficient to describe the structures during the reaction in the crystal. This is not easy requirement because of fast catalytic reaction-rate of the enzyme, msec-mu sec. To restrict the catalytic reaction-rate, we prepared some mutants by site-directed mutagenesis. The Km value of R210K mutant was similar to that of the wild-type, but its kcat value drastically decreased. Laue data of R210K by synchrotron radiation were processed using the program LAUEMAD. The power and limitations of time resolved X-method by using Laue diffraction were investigated. In particular, the effects of crystalline disorder, imperfect wavelength scaling, and a systematic lack of completeness in Laue structure factor sets at low and medium resolution were analyzed.As results of the above investigation, we learned that R210K would be a good target of time resolved X-ray structure study combined Laue and monochromatic diffraction techniques.