A simple approach to identify the mechanism of intermediate transfer: enzyme system related to triose phosphate...

A new approach is described to identify the mechanism of transfer of intermediates of consecutive reactions catalysed by two functionally related enzymes. Interactions resulting in conformational changes of the individual enzymes and/or channelling of the intermediate can be identified by comparing the rate constants of the coupled and individual reactions. Using these kinetic parameters, the relative specific radioactivity of the end product can be calculated according to the different mechanisms. The comparison of these values with the experimentally determined relative specific radioactivity enhances the sensitivity of the determination. The interaction between aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) and glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) was analysed. The data agree with the model in which channeling of the intermediate was assumed. The results suggest that glyceraldehyde 3-phosphate is functionally compartmentalised within the reconstituted enzyme system, which may be relevant under physiological conditions.