Involvement of Arginine 143 in Nucleotide Substrate Binding at the Active Site of Adenylosuccinate Synthetase from Escherichia coli

Abstract

Adenylosuccinate synthetase from Escherichia coli is inactivated in a biphasic reaction by guanosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)thio]phosphate (GMPSBDB) at pH 7.1 and 25 degrees C. Reaction of the enzyme with [8-3H]GMPSBDB results in the incorporation of 2 mol of the reagent/mol of subunit; in the presence of active site ligands the incorporation is reduced to 1 mol of reagent/mol of subunit. GMPSBDB reacts with Cys-291 in the initial rapid reaction which is accompanied by loss of 50% of the enzymatic activity; this reaction is not affected by the presence of active site ligands. In the slower reaction, GMPSBDB inactivates the enzyme by reacting with Arg-143. The inactivation kinetics of the slower phase are consistent with the formation of an enzyme--GMPSBDB complex having a Kd of 42 microM. Active site nucleotides, either adenylosuccinate or IMP + GTP, prevent both slower phase inactivation and labeling of Arg-143. Replacement of Arg-143 with a Leu by site-directed mutagenesis does not change the catalytic constant or the K(m) for aspartate but does significantly impair nucleotide binding: the Michaelis constants for IMP and GTP increase by 60-fold and 10-fold, respectively, in the R143L mutant. The crystal structure of the E. coli enzyme [Poland, B.W., Silva, M.M., Serra, M.A., Cho, Y., Kim, K. H., Harris, E.M.S., & Honzatko, R.B. (1993) J. Biol. Chem. 268, 25334--25342] shows that Arg-143 from one subunit projects into the putative active site of the other subunit. These results indicate that both subunits of dimeric adenylosuccinate synthetase contribute to each active site and that Arg-143 plays an important role in nucleotide binding.

Variations in the Response of Mouse Isozymes of Adenylosuccinate Synthetase to Inhibitors of Physiological Relevance

“…Native states of oligomerization for each isozyme remain ambiguous, as reports of monomeric and dimeric AdSS1 and AdSS2 are in the literature (8, 10, 29 -31). In contrast, the synthetase from Escherichia coli is active as a dimer (32,33) and exists in a monomer-dimer equilibrium (34).…”

Chemical Modification and Site-directed Mutagenesis of Conserved HXXH and PP-loop Motif Arginines and Histidines in the Murine Bifunctional ATP Sulfurylase/Adenosine 5′-Phosphosulfate Kinase

“…Histidines have been implicated in metal ion binding (27) and electrostatic stabilization of intermediates (28). The catalytic importance of arginine has been shown in nucleotide substrate binding (29), and it may form a catalytic triad with serine and asparagine (30). Arginine can also have structural importance, as demonstrated by its role in conformational changes in murine leukemia virus reverse transcriptase (31).…”

A Study of Escherichia coli Adenylosuccinate Synthetase Association States and the Interface Residues of the Homodimer

“…Slow phase inactivation of AMPSase was observed when guanosine-5Ј-O-[S-(4-bromo-2,3-dioxobutyl)] thiophosphate modifies the enzyme at Arg 143 . Modification of Arg 143 is prevented by adenylosuccinate alone or by a mixture of GTP, MgCl 2 , and IMP (14).…”