18] 5-Phosphoribose pyrophosphokinase: Ribose 5-phosphate + ATP → PRRP + AMP

Abstract

Publisher Summary This chapter describes the assay method and properties of 5-phosphoribose pyrophosphokinase. The reaction is assayed by the convenient two-step procedure of Kornberg. In step I, the pyrophosphokinase reaction is carried out leading to the accumulation of 5-phosphoribosylpyrophosphate (PRPP), and in step II the accumulated PRPP is quantitatively utilized for the enzymatic conversion of orotate to a mixture of orotidylate and UMP. The removal of orotate in step II is determined spectrophotometrically. Alternatively, two other two-step procedures of greater sensitivity (but less convenience) are available. These involve the enzymatic conversion of C-adenine and 5-amino-4-imidazolecarboxamide to their respective ribotides. The assay method has been successfully applied to a wide variety of crude tissue extracts. The enzyme appears to be specific for ATP and ribose 5-phosphate. ADP, ITP, and UTP, at concentrations equal to that of the ATP used in the assay, either failed to replace ATP or showed less than 2% of the activity of ATP. Similarly, ribose 1-phosphate, 2-deoxyribose 5-phosphate, and glucose 6-phosphate failed to replace ribose 5-phosphate. 2-Deoxyribose 5-phosphate in the presence of an equimolar concentration of ribose 5-phosphate did not inhibit the formation of PRPP.