Yeast 3-phosphoglycerate kinase is inactivated by incubation with pyridoxal 5'-diphospho-5'-adenosine (AdoP2Pxy) [Tamura, J. K., Rakov, R. D. & Gross, R. L. (1986) J. Biol. Chem. 261, 4126-4133). Incorporation of 1 mol affinity label/mol enzyme was sufficient for complete inactivation of 3-phosphoglycerate kinase. The substrate ATP affords substantial protection against inactivation. Partial protection is afforded by the substrate glycerate 3-phosphate. When AdoP2Pxy-modified phosphoglycerate kinase was reduced with [3H]NaBH4 and subjected to trypsin hydrolysis, only one radioactive peptide was isolated by reverse-phase high-performance liquid chromatography. The amino acid composition and sequence analysis of the purified radioactive peptide revealed that it spans residues 379-403 of the enzyme and Lys385 specifically reacted with the affinity label. This peptide represents the hinge region between the two domains of the protein, where the active site is also located. The fluorescence intensity of enzyme-bound AdoP2Pxy is enhanced when glycerate 3-phosphate is added, suggesting exposure of the fluorescent probe to a more hydrophobic environment. Another fluorescent analog, anthraniloyl-dATP (ant-dATP), which carries the fluorescent reporter group on the ribose ring, binds to the enzyme at two distinct sites with Kd values of 6 +/- 2 microM and 25 +/- 3 microM, as determined by steady-state anisotropy measurements. Bound ant-dATP was displaced from the enzyme by glycerate 3-phosphate and ATP, as monitored by the fluorescence anisotropy. These results suggest that both fluorescent ATP analogs bind to the active site, which is at the hinge region of the enzyme. Model-building studies showed that when AdoP2Pxy is built into the open form of the enzyme, as described in X-ray studies, the pyridoxyl group of AdoP2Pxy cannot reach Lys385 for Schiff-base formation. Labeled Lys385 is on a beta-turn immediately following helix XII, which was suggested to interact with the nucleotide and become ordered at the active site of 3-phosphoglycerate kinase [Watson, H. C., Walker, N. P. C., Shaw, P. J., Bryant, T. N., Wendell, P. L., Fothergill, L. A., Perkins, R. E., Conroy, S. C., Dobson, M. J., Tuite, M. F., Kinesman, A. J. & Kinesman, S. M. (1982) EMBO J. 1, 1635-1640]. The results presented here suggest that binding of substrates cause significant structural changes in the enzyme.