[28c] Phosphoribulokinase

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This chapter describes the assay method, purification procedure, and properties of phosphoribulokinase. For routine enzyme assay, ribose 5-phosphate is the substrate and an excess of phosphoriboisomerase is added. Under these conditions, the rate of reaction can be followed by the appearance of alkali-labile phosphate, since both phosphate groups of ribulose diphosphate are alkali-labile, 2 and ribose 5-phosphate, ATP, and ADP do not yield inorganic phosphate. An alternative assay involved the determination of ADP with phosphoenolpyruvic kinase and lactic dehydrogenase. The final preparation is without any activity on the following substrates: D-xylulose 5-phosphate, fructose 6-phosphate, and sedoheptulose 7-phosphate. Ribose 5-phosphate is phosphorylated very slowly, but this is due to the presence of phosphoriboisomerase in the final enzyme preparation. In the absence of sulfhydryl compounds, the enzyme is irreversibly inactivated after repeated freezing and thawing. The enzyme is inactivated by the addition of Hg⁺⁺ and p-ehloromercuribenzoate. These effects are reversed by the addition of cysteine. The phosphorylation reaction proceeds at maximal velocity at pH 7.9. The enzyme is suited for the quantitative determination of ribulose 5-phosphate, although it is not completely specific, since, with the relatively large quantities of enzyme preparation required, ribose 5- phosphate also reacts.