Soluble, bifunctional enzyme aggregates have been prepared by crosslinking sequentially acting enzymes with glutaraldehyde. Aggregates of j3-glucosidase and glucose oxidase with molecular weights in the range 200 000-300 000 have been characterized from such a preparation. The sequentially working enzymes malate dehydrogenase and citrate synthase have also been aggregated using the same method. The kinetic behaviour of a system comprising two sequentiaily acting enzymes, either separately or as a n aggregate, was studied in media containing increasing concentrations of poly(ethyleneglyco1). A plot of transition time against polymer concentrations was bell-shaped in the case of /3-glucosidase and glucose oxidase whereas in the system malate dehydrogenase-citrate synthase almost no lagphase was observed and comequently no transition time was registered. I n both enzyme systems the steady-state rate in the overall reaction was stimulated up to 30 O/ , , a t lower polymer concentrations, whereas reduced rates were obtained in more concentrated polymer solutions. These effects are interpreted in terms of (a) an exclusion effect leading to a relative enrichment of the rate-limiting intermediate and an apparently higher concentration of the enzymes in the sequence and (b) a decreased diffusion rate which lowers the apparent V.
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