Involvement of arginine 143 in nucleotide substrate binding at the active site of adenylosuccinate synthetase from Escherichia coli.
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.
Moe
O A
OA
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716, USA.
Baker-Malcolm
J F
JF
Wang
W
W
Kang
C
C
Fromm
H J
HJ
Colman
R F
RF
D000265:Adenylosuccinate Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D007291:Inosine Monophosphate / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008666:Metalloendopeptidases; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000378:metabolism* / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D016415:Sequence Alignment; D013873:Thionucleotides / Q000378:metabolism* / Q000494:pharmacology
D000345:Affinity Labels; D004791:Enzyme Inhibitors; D008024:Ligands; D009711:Nucleotides; D010455:Peptides; D013873:Thionucleotides; C086303:guanosine 5'-O-(S-(4-bromo-2,3-dioxobutyl))thiophosphate; D007291:Inosine Monophosphate; D006157:Guanosine Monophosphate; D001120:Arginine; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus; D000265:Adenylosuccinate Synthase; D003545:Cysteine