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Metabolism and Enzymology

Isolation of Chloroplastic Phosphoglycerate Kinase ¹

Kinetics of the Two-Enzyme Phosphoglycerate Kinase/Glyceraldehyde-3-Phosphate Dehydrogenase Couple

Department of Biological Sciences (m/c 066), University of Illinois at Chicago, Box 4348, Chicago, Illinois 60680, Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802

We report here a method for the isolation of high specific activity phosphoglycerate kinase (EC 2.7.2.3) from chloroplasts. The enzyme has been purified over 200-fold from pea (Pisum sativum L.) stromal extracts to apparent homogeneity with 23% recovery. Negative cooperativity is observed with the two enzyme phosphoglycerate kinase/glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) couple restored from the purified enzymes when NADPH is the reducing pyridine nucleotide, consistent with earlier results obtained with crude chloroplastic extracts (J Macioszek, LE Anderson [1987] Biochim Biophys Acta 892: 185-190). Michaelis Menten kinetics are observed when 3-phosphoglycerate is held constant and phosphoglycerate kinase is varied, which suggests that phosphoglycerate kinase-bound 1,3-bisphosphoglycerate may be the preferred substrate for glyceraldehyde-3-P dehydrogenase in the chloroplast.

¹ This research was supported by grants from the U.S. Department of Energy (DE-FG02-85-ER 60367) and from the University of Illinois, Chicago Research Board.