97] Bacterial ribosephosphate isomerase

Abstract

This chapter discusses the several assay procedures available for detecting ribosephosphate isomerase activity. Ribosephosphate isomerase (RPI) catalyzes the interconversion of D-ribose 5-phosphate and ribulose 5-phosphate. The enzyme is found in both heterotrophic and autotrophic bacteria and its perceived role in metabolism differs significantly depending on whether it is considered to be an anabolic or a catabolic enzyme. In heterotrophic organisms, it functions catabolically as a member of the pentose pathway. The pentose shunt in heterotrophs is primarily involved in the production of nicotinamide adenine dinucleotide (NADPH) and for supplying ribose 5-phosphate for the formation of 5-phosphoribosyl pyrophosphate and the subsequent synthesis of nucleic acids. In autotrophic bacteria, the enzyme occupies a position in the anabolic pentose pathway, which serves as the major source of reduced carbon for these organisms. The procedure is appropriate at all stages of enzyme purification and can be used for kinetic studies as well. Direct spectrophotometric assays are also described in the chapter in which the absorbance of ribulose is monitored at 280 nm or at 290 nm. The final stages of purification are most often accomplished by ion-exchange chromatography, gel filtration, or affinity chromatography.