The interaction of FBPase with aldolase: a kinetic and fluorescence investigation on chicken muscle enzymes

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) is strongly inhibited by AMP in vitro and, therefore, at physiological concentrations of substrate and AMP, FBPase should be completely inhibited. Desensitization of rabbit muscle FBPase against AMP inhibition was previously observed in the presence of rabbit muscle aldolase. In this study, we analysed the kinetics of an FBPase catalyzed reaction and interaction between chicken muscle FBPase and chicken muscle aldolase. The initial rate of FBPase reaction vs. substrate concentration shows a maximum activity at a concentration of 20 muM Fru-1,6P(2) and then decreases. Assuming rapid equilibrium kinetics, the enzyme-catalyzed reaction was described by the substrate inhibition model, with K-s approximate to 5 muM and K-si approximate to 39 muM and factor beta approximate to 0.2, describing change in the rate constant (k) of product formation from the ES and ESSi complexes. Based on ultracentrifugation studies, aldolase and FBPase form a hetero-complex with approximately 1:1 stoichiometry with a dissociation constant (K-d) of 3.8 muM. The FBPase-aldolase interaction was confirmed via fluorescence investigation. The aldolase-FBPase inter-action results in aldolase fluorescence quenching and its maximum emission spectrum shifting from 344 to 356 nm. The K-d of the FBPase-aldolase complex, determined on the basis of fluorescence changes, is 0.4 muM at 25 degreesC with almost 1:1 stoichiometry. This interaction increases the I-0.5 for the AMP inhibition of FBPase threefold, and slightly affects FBPase affinity to magnesium ions, increasing the K-a and Hill coefficient (n). No effect of aldolase on the FBPase pH optimum was observed. Thus, the decrease in FBPase sensitivity to AMP inhibition enables FBPase to function in vivo thanks to aldolase. (C) 2003 Elsevier Inc. All rights reserved.