A possible in vivo mechanism of intermediate transfer by glycolytic enzyme complexes: steady state fluorescence anisotropy analysis of an enzyme complex formation.

Rate constants of dissociation (k(off)) and association (k(on)) of the bienzyme complex yeast glyceraldehyde-3-phosphate dehydrogenase--yeast alcohol dehydrogenase have been determined in the absence and presence of NAD or NADH by fluorescence anisotropy measurements. We found that dissociation of the complex is considerably slower than catalytic turnover of either of the enzymes (that is k(off) much less than kcat) irrespective of the presence of coenzymes. A perusal of the literature reveals that this relation invariably applies to all systems studied so far. These observations all taken together constitute compelling evidence that direct metabolite transfer in enzyme complexes cannot be satisfactorily described by invoking the dynamic model but requires a model assuming more lasting complexes. This seems to support the case of the temporary-stationary model suggested by one of us. Implications of this conclusion are treated in depth and further evidence is cited under Discussion.