The binding of bromophenol blue to aspartate aminotransferase

Abstract

Several experiments are presented that suggest that the sulfonphthalein dye, bromophenol blue, binds at, or near to, the active site of pig heart extramitochondrial aspartate aminotransferase (EC 2.6.1.1.). The binding is characterized at pH 8.0 by a bathochromic shift in the dye's visible spectrum from 590 to 599 nm. Changes in absorption at 612 nm are used to calculate dissociation constants for the enzyme-dye complex. These constants are a linear function of buffer concentration, and the dissociation constant in the absence of buffer is calculated to be 0.6 × 10 −6 m. Acetate and glutarate are shown to be competitive inhibitors of the dye's binding. However, similar experiments and kinetic data indicate that the substrates, erythro-β-hydroxy- l-aspartate and l-aspartate, displace the dye in an uncompetitive fashion. Direct spectroscopic evidence proves the existence of a ternary complex of bromophenol blue, aspartate aminotransferase, and erythro-β-hydroxy- l-aspartate. Rapid reaction kinetic experiments further show that the dye may be displaced by erythro-β-hydroxy- l-aspartate through formation of this ternary complex. Addition of 1.9 m sucrose to a solution of the aspartate aminotransferase-bromophenol blue complex causes a 2-nm bathochromic shift in the bound dye's visible spectrum; the free dye has a 5-nm bathochromic shift under the same set of conditions. It is concluded that the dye molecule binds through multiple interactions at the protein surface. Such multiple interaction loci can explain the differences observed in the way that substrates and inhibitors displace the dye molecule from the enzyme.