Preparation of homogeneous elongation factor G and examination of the mechanism of guanosine triphosphate hydrolysis.

A new procedure for the isolation of physically and enzymatically homogeneous Elongation Factor G based on the isolation of an Elongation Factor G. ribosome . GDP. fusidic acid complex is described. The yield from this procedure is 23 to 36% of the theoretical and results in an enzyme of specific activity 6200 to 6700 units per AyaO. The enzyme was shown to be physically homogeneous by disc gel electrophoresis under both native and denatured conditions. The enzymatic purity of the preparation with respect to enzymes utilizing guanine nucleotides was examined by quantitation of contaminating nonribosomal dependent GTPase, GDPase, nucleoside diphosphate kinase, and polynucleotide phosphorylase activities. The highest contaminating activity was found to be 0.002% of the activity of Elongation Factor G. Utilizing this homogeneous Elongation Factor G, the reaction involving the hydrolytic cleavage of GTP catalyzed by the ribosome and Elongation Factor G was examined. The position of GTP cleavage was elucidated by the technique of I80 incorporation from water. It was demonstrated that solvent oxygen is incorporated only into the inorganic phosphate derived from the y-phosphate of GTP. The number of solvent oxygen atoms incorporated per phosphate was 0.81 + 0.19. Neither the (Ynor P-phosphates of GDP, the other product of hydrolysis, incorporated solvent oxygen. From these observations the position of GTP cleavage was identified as occurring between the y phosphorus atom and the oxygen bridging the p and y phosphorus atoms. Using Wi and [cY-~~P]GDP, it was determined that neither the exchange of Pi into GTP nor the exchange of GDP into GTP are catalyzed by Elongation Factor G and the ribosome.