Add time:08/04/2019         Source:sciencedirect.com

Publisher SummaryThis chapter describes the synthesis of ribulose 1,5-bisphosphate. The synthesis of ribulose 1,5-bisphosphate (RuBP) is accomplished by two routes: (1) glucose 6-phosphate and (2) adenosine monophosphate. These routes provide procedures suitable for preparing several hundred grams of RuBP and of the major intermediates lying between starting materials and this metabolite. The enzymes required are all used in an immobilized form. Both routes require ATP cofactor recycling and the procedure includes the details of an improved synthesis of the acetyl phosphate used as the ultimate phosphate donor for this recycling. The route based on glucose 6-phosphate (G-6-P) also illustrates a convenient procedure for the regeneration of nicotinamide adenine dinucleotide phosphate [NAD(P)] from nicotinamide adenine dinucleotide phosphate dehydrogenase [NAD(P)H] under anaerobic conditions, based on the conversion of α-ketoglutarate to glutamate. It is important to minimize the exposure of the RuBP to isomerizing conditions during and after preparation. The procedures for the preparation of RuBP are also summarized in the chapter. Glucose 6-phosphate is prepared using hexokinase-catalyzed phosphorylation and adenosine triphospahte (ATP) regeneration. Two methods employed for RuBP determination are the coupled-enzyme and radiometric method.

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