In Vivo Activity, Purification, and Characterization of Shikimate Kinase from Sorghum

Abstract

Sorghum bicolor stems and shoots were observed to phosphorylate exogenously supplied shikimate, forming a product which could not be distinguished from shikimate 3-phosphate by anion exchange and thin layer chromatography. Upon treatment with phosphatase, this product gave rise to a compound which co-chromatographed with shikimate. Shikimate kinase from stems was separated from phosphatase and ATPase and partially characterized. The stoichiometry of the reaction required equimolar quantities of ATP and shikimate to produce ADP and shikimate 3-phosphate. Maximal enzymic activity was observed near pH 9 in the presence of 11 millimolar MgCI2. Graphs of reaction velocity as a function of substrate concentration were hyperbolic for both substrates and Km values of 0.2 and 0.11 millimolar were calculated for shikimate and ATP, respectively. Shikimate kinase was not inhibited by phenylalanine, tyrosine, or tryptophan, either alone or in combination. Slight inhibition was caused by p-coumarate and greater inhibition by caffeate. Inhibition was also observed in the presence of ADP and AMP, but the reaction velocity was not highly responsive to adenylate "energy charge" in experiments with ADP-ATP mixtures. Plants and microorganisms are known to synthesize aromatic amino acids by the shikimate pathway (30). One step in the pathway in microorganisms is the phosphorylation of shikimate. The enzyme which catalyzes that reaction, shikimate kinase (EC 2.7.1.71 ATP:shikimate 3-P-transferase) has been reported to be present in cell-free preparations of microorganisms and lower plants (4, 11, 13). We recently found shikimate kinase in cell-free preparations of etiolated shoots and mature green stems of Sorghum (7), and Koshiba (16) subsequently detected the enzyme in preparations of Phaseolus mungo. However, the partial purification and characterization of shikimate kinase and the in vivo phosphorylation of exogenously supplied shikimate have not been described. The flow of C through the shikimate pathway to the major branch point chorismate is controlled in microorganisms by feedback inhibition and repression of D-arabinoheptulosonate-7-P synthetase (21) and, in Bacillus subtilis, of shikimate kinase (13). Such phenomena have not been demonstrated in green plants (14, 28) and the mechanisms which regulate the formation of choris-mate are unknown. Green plants may differ from microorganisms in providing alternate fates for shikimate and its precursors (26, 27). Shikimate kinase, catalyzing an irreversible reaction in the '