Glycolytic enzyme inhibition by Tartronic Acid Phosphate and its derivatives




Thomas, Marian K.

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Tartronic Acid Phosphate (TAP) has been synthesized in a single-step reaction by permanganate oxidation of [beta]-glycerol phosphate followed by selective crystallization as the cyclohexylammonium salt. TAP was designed to be an analog of 2-phosphoglyceric acid and a precursor of TAP methyl ester, hydrazide and azide derivatives which were potential affinity labels with specific regard to the glycolytic enzymes enolase, phosphoglycerate mutase, and pyruvate kinase. Several other methods of TAP synthesis were tried: phosphorylation of tartronic acid by POCl[lowered 3] under a variety of conditions yielded complex mixtures of products and bromine oxidation of glycerol phosphate yielded primarily glyceric acid 2-phosphate. TAP was found to competetively inhibit the three enzymes mentioned above with Ki's of 7.27xlO[raised -4]M, 3.1xl0[raised -5]M and 6.4x10[raised -5]M, respectively. In addition, it was found to be a poor competetive inhibitor of glyceraldehyde 3-phosphate dehydrogenase, Ki = 10[raised -2]M, and had no effect on 3-phosphoglycerate kinase or triosephosphate isomerase. Further studies with TAP included its use as an affinity column ligand to selectively purify the enzymes which it inhibited; however, such columns made were not successful. TAP dimethyl ester and dihydrazide had no effect on enolase or PG mutase; the dihydrazide irreversibly inhibited pyruvate kinase. TAP diazide had no effect on PG mutase, but did exhibit a biphasic pattern of irreversible inactivation of enolase. A rapid decrease in enzyme activity was first observed, followed by a lag period in which enolase activity did not further decrease until the enzyme was rechallenged by by the addition of more diazide. Some protection against inhibition was afforded by the substrate 2PGA. When pyruvate kinase was tested with TAP diazide, it was found that nitrite ion, used to generate the diazide from acidic dihydrazide, inhibited the enzyme at a level of 10[raised -3]M or greater. The compound bromoacetyl ethanolamine phosphate was found to completely and irreversibly inhibit PG mutase while not affecting enolase and was used to inactivate that enzyme when mutase-free preparations of enolase were needed for kinetic studies.