SITE-DIRECTED MUTAGENESIS SHOWS THAT LYSINE-299 IS ESSENTIAL FOR ACTIVITY OF PEA CHLOROPLAST FRUCTOSE-1,6-BISPHOSPHATASE

Lysine-299 of the pea (Pisum sativum L. cv. Lincoln) chloroplast fruct ose-1,6-bisphosphatase (FBPase, EC 3.1.3.11) was substituted by glycin e, glutamic acid and valine by codon mutation of specific nucleotides, and PCR construction using as target a cDNA clone coding for the pea photosynthetic enzyme. The mutagenized Escherichia coil-expressed FBPa ses showed K-m values in the same order of magnitude as that of the wi ld type-expressed enzyme. On the contrary, the V-max of the glutamic a cid mutant was fourfold lower than that of the wild type FBPase, where as the glycine and valine mutants exhibited an intermediate behaviour. These results contrast with those reported for the gluconeogenic FBPa se from rat liver, in which a mutation of lysine-274 (the homologous c ounterpart for lysine-299 of the pea chloroplast enzyme) to alanine pr oduced a sharp increase in the K-m value (M.R. El-Magrabi et al., Jour nal of Biological Chemistry 267 (1992) 6526-6530). We therefore conclu de that, as observed with the cytosolic FBPases, lysine-299 of the pea plastidic enzyme plays an essential role in FBPase activity. However, unlike the case with the cytosolic enzyme, this lysine appears more c oncerned with the normal catalytic process than with substrate binding .