Fluorescent labelling of pyruvate carboxylase with 4-acetamido-4'-(iodacetyl)-aminostilbene-2,2'-disulfonic acid (AIDA) provides a means of monitoring substrate binding.

Pyruvate carboxylase [EC 6.4.1.11 is a biotindependent enzyme that catalyses the carboxylation of pyruvate to form oxaloacetate (for a review see [ 11). The overall reaction proceeds in two stages, in the first stage the biotin prosthetic group is carboxylated by H Q with concomitant cleavage of MgATP. The carboxybiotin then moves to another part of the active site where the carboxyl group is transferred to pyruvate. In addition to the substrates, the reaction also requires free Mg2+ and acetyl CoA is an activator without which, the chicken liver enzyme shows little activity [2]. The chicken liver enzyme used in the experiments described here comprises of four identical subunits of molecular weight = 125 kDa. Each subunit contains one molecule of covalently bound biotin. In the past, efforts have been made to study substrate and effector binding using dye displacement [3] or the fluorescent analogue of ATP i.e. formycin A-5'-mphosphate F P ) [4]. A detailed kinetic study of nucleotide binding was recently carried out using FP [5 ] . Two drawbacks of this approach are that owing to the fluorescence inner-filter effect, near saturation of the enzyme with FlT cannot be achieved and the problems inherent in using a structural analogue as opposed to the actual substrate. A way of enabling the study of substrate and effector binding to be carried out is to covalently label the enzyme in such a way that the label reports the binding. AIDA is a water-soluble, fluorescent, sulphydryl-modifying reagent (Molecular Probes). Chicken liver pyruvate carboxylase was labelled with AIDA by incubating the fluorescent reagent and the enzyme in 1:l molar proportions (in terms of biotin content of the enzymes assayed as described previously [6]). After 30 min incubation, the extent of labelling achieved was 0.96 AIDA molecules per biotin. From the timecourse of the reaction, labelling appeared to occur in a single process. Any unreacted AIDA was then removed by passage of the reaction mixture through Sephadex G-25 as described previously [7]. During the course of the labelling, enzymic activity was monitored with a spectrophotomemc assay to detect oxaloacetate formation [8]. At the point where the extent of labelling was 0.96, the enzyme was found to have retained 67% of its activity relative to a control incubated in the absence of ADA. As can be seen in Fig. 1, pyruvate induces quenching of fluorescence across the spectrum, however there is no marked