Investigation of the pre-steady-state kinetics of fructose bisphosphatase by employment of an indicator method

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Abstract
The pre-steady-state kinetics for the hydrolysis of fructose 1,6-bisphosphate by rabbit liver fructose bisphosphatase were investigated by stopped-flow kinetics utilizing an acid-base indicator method that permits the continuous monitoring of the inorganic phosphate product. The reaction sequence is characterized by 2 successive 1st-order steps followed by establishment of the steady-state rate. The 1st exponential process results form a conformational change in the protein that is dye sensitive owing to a perturbation of an acidic residue on the protein. A 2nd process reflects the rapid initial turnover of all 4 subunits of the enzyme with the concomitant release of inorganic phosphate followed by the rate-limiting step of the catalytic cycle. This latter step may involve a product release (fructose 6-phosphate) or a 2nd conformational change. The catalytic cycle ends with decay of the enzyme to its initial unreactive resting state.