Enzymes of Pentose Biosynthesis: I. Separation of Pentose Cycle Enzymes of Escherichia Coli

Abstract

A study of the enzymes of the pentose cycle in Escherichia coli has been undertaken. Procedures are reported for separating the cell extract into fractions containing transkeblase, ~-xylulose-5-phosphate-3-epimerase, and transaldolase and phosphoribose isomerase. Cross contamination was small enough and yields of enzymatic activity were adequate to make the fractions usable for further purification steps. Preliminary results of the further purificatio~m of the enzymes, some properties of the enzymes, and a number 0% problems in enzyme assay are discussed.

-Ribose-5-Phosphate Isomerase from Spinach: Heterologous Overexpression, Purification, Characterization, and Site-Directed Mutagenesis of the Recombinant Enzyme

“…The reaction mixture (1 ml) contained 40 mM KCl, 10 mM MgCl 2 , 1 mM ATP, 3 mM PEP, 0.24 mM NADH, 2 mM R5P, 2 units of phosphoribulose kinase, 4 units of pyruvate kinase, 5 units of lactate dehydrogenase, and 0.01 to 0.1 unit of RPI in 50 mM Bicine buffer, pH 8.0. In order to measure the reverse reaction, RPI activity was coupled via transketolase, TIM, and glycerol phosphate dehydrogenase (28). The reaction mixture (0.2 ml) at 25°C contained 10 mM MgCl 2 , 1 mM EDTA, 1 mM DTT, 0.1 mM thiamine pyrophosphate, 0.25 mM NADH, 1 mM Ru5P, 5 mM xylulose 5-phosphate, 0.2 units of transketolase, 4 units of TIM, 0.4 units of glycerol phosphate dehydrogenase, and 0.002 to 0.02 unit of RPI in 50 mM Bicine buffer, pH 8.0.…”