Purification and Properties of NADP dependent Non phosphorylating Glycer-aldehyde 3 phosphate Dehydrogenase from the Green Alga Chlamydomonas reinhardtii

A.A. IGLESIAS, A. SERRANO, M.G. GUERRERO, M. LOSADA

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS

Año: 1987 vol. 925 p. 1 - 10

0304-4165

NADP-dependent non-phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (]EC 1.2.1.9), previouslydescribed in higher plants, has been now found to be present in eukaryotic green algae, but in neithercyanohacteria nor non-photosynthetic microorganisms. The enzyme from the unicellular green algaCIdamydomonas reinhardtii, strain 6145c, has been purified to apparent electrophoretic homogeneity. Thenon-phosphorylating enzyme was effectively separated from the NADP-dependent phosphorylating 13-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) by dye-ligand chromatography on Reactive Red-120agarose. The purified enzyme exhibited an optimum pH in the 8.5-9.0 range and a specific activity ofapprox. 8/tmol. (mg protein) -1 -min -l. The native protein was characterized as a homotetramer with amolecular weight of 190000, a Stokes radius of 5.2 mn, and an isoelectric point of 6.9. From kinetic studies,Kin-values of 9.8 and 51/~M were calculated for NADP and D-glyceraldehyde 3-phosphate, respectively, anabsolute specificity for both substrates being observed. L-Glyeeraldehyde 3-phosphate was a potent non-competitiveinhibitor (Ki, 48/tiM). The reaction products NADPH and D-3-phosphoglycerate inhibited enzymeactivity in a competitive manner with respect to NADP (Ki, 78 pM) and D-glyceraldehyde 3-phosphate (Ki,1.2 mM), respectively. Thermal inactivation occurred above 45"C and was effectively prevented by eithersubstrate. The presence of essential vicinal thiol groups is suggested by the inactivation produced bydiamide, with D-glyceraldehyde 3-phosphate, but not NADP, behaving as a protective agent. The enzyme´spossible physiological role in photosynthetic metabolism is discussed briefly.