Specificity of pyruvate kinase.

Short Communications Specificity of pyruvate kinase Previous reports have indicated that pyruvate kinase is a relatively non-specific enzyme capable of catalyzing the transfer of phosphate from PEP to a wide variety of nucleoside diphosphate acceptors l& In the present studies, attempts were made to utilize this enzyme for the assay of UDP.The rate of phosphate transfer to nucleoside diphosphate was followed by coupling reactions II (pyruvate kinase) and III (lactic dehydrogenase) and measuring the change in absorbance at 340 m/* indicative of the oxidation of DPNH. Preliminary results indicated that the assay for UDP was variable and was dependent on the source of the nucleotide, and the age and purity of the enzyme preparations employed for assay purposes. Since the possibility remained that reaction I (nucleoside diphosphokinase 3) was being coupled to reactions II and III, the specificity of pyruvate kinase was examined. Pyruvate kinase was prepared as described and twice crystallized 4. The crystalline preparation was diluted I to 5o before use giving a protein concentration of 8/*g per aliquot used in the spectrophotometer cuvette. Lactic dehydrogenase was obtained from a commercial source; DPNH and the nucleotides were products of the Sigma Chemical Co., except as indicated. Routine assays were conducted by the addition of pyruvate kinase to appropriate cuvettes containing the following in a final volume With a sample of purified UDP*, an apparent lag period occurred before the oxidation of the DPNH (Fig. I). Preincubation of this sample with pyruvate kinase but without PEP, followed by the addition of PEP to initiate the reaction, resulted in DPNH oxidation at a rate comparable to that observed when ADP was acting as a substrate. These results indicated that a reaction other than the direct phosphorylation of the UDP by the PEP was taking place. This reaction, I, catalyzed by nucleoside diphosphokinase, requires the presence of only trace amounts of adenine nucleotides as contaminants of the materials under study to generate sufficient ATP via reaction II so that the rate of reactions I and II increases in an autocatalytic fashion. Chromato-graphic analysis of commercial UDP indicated the presence of approximately 5 % of