T. Nowak, A. Mildvan
Nov 25, 1970
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Journal
The Journal of biological chemistry
Abstract
Abstract The optically pure d and l isomers of 2-phospholactate were synthesized by phosphorylation of the appropriate lactic acids. The interactions of these analogues and of phosphoglycolate with several enzymes that catalyze reactions of phosphoenolpyruvate were examined by kinetics and by direct binding studies measuring the longitudinal relaxation rate of water protons. With pyruvate kinase, the Ki of d-phospholactate (21 µm) and of phosphoglycolate (52 µm) agree with the Km of phosphoenolpyruvate (26 µm), while the Ki of l-phospholactate (380 µm) is 18-fold greater. This difference in affinity is confirmed in the direct binding studies. The enhancement of the ternary pyruvate kinase-Mn-inhibitor complex of d-phospholactate (et = 1.9) and of phosphoglycolate (et = 1.7) agree with that of phosphoenolpyruvate (et = 2.2), but differs from that of l-phospholactate (et = 3.8), indicating a structural difference in this latter metal bridge complex. With enolase, the inhibition was not stereospecific since the Ki values of both isomers of phospholactate and of phosphoglycolate were equal (370 µm) and larger than the Km of 2-phosphoglycerate (6.7 µm). Equal affinities for the analogues were confirmed by direct binding studies, but unequal ternary enhancements were observed for the substrate phosphoenolpyruvate (et = 3.7), d-phospholactate (et = 11.6), phosphoglycolate (et = 8.4), and l-phospholactate (et = 5.8) because of structural differences in their respective ternary complexes. Phosphoenolpyruvate carboxykinase, which catalyzes a reaction with a mechanism homologous with that of pyruvate kinase, shows the opposite inhibition pattern since it is preferentially inhibited by l-phospholactate (Ki = 28 µm) as compared with d-phospholactate (Ki = 292 µm). None of the analogues inhibited the reaction catalyzed by phosphoenolpyruvate synthetase. The patterns of inhibition by the phospholactates of pyruvate kinase (d >> l), enolase (d = l), and carboxykinase (l >> d) are explained in terms of the known stereochemistries of the reactions catalyzed by these enzymes and the structures at their respective active sites.