Effects of ATP and CTP on the conformation of the regulatory subunit of Escherichia coli aspartate transcarbamylase in solution: a medium-resolution hydrogen exchange study.
Medium-resolution hydrogen exchange methods have been used to examine the solvent accessibility of seven peptides in the regulatory subunit (r2) of Escherichia coli aspartate transcarbamylase in the presence and absence of ATP and CTP. Both nucleotides are allosteric effectors of the holoenzyme; binding of ATP increases the affinity of the holoenzyme for the substrate L-Asp, while CTP has the opposite effect. Following Rosa and Richards (1979, 1981, 1982) and Englander et al. (1983, 1985), exchange-out curves for individual peptides were generated by adjusting the pH to 2.7 to quench exchange-out, digesting the protein with pepsin, separating peptides by reverse-phase HPLC, determining their radioactivity, and correcting for radioactivity lost during the analysis. Sixteen peptides from segments 1-11 and 76-153 were identified by amino acid and N-terminal analysis. Nine fell in regions where background was too high or were present at too low concentrations for exchange to be monitored. The number of protons whose exchange could be followed in peptides 1-11, 76-91, 78-90, 84-101, 93-112, 108-114, and 115-125 ranged from approximately 1 (1-11, 108-114) to 10 (84-101) and 11 (93-112). The pattern of results obtained suggests that the structure of r2 in solution is similar to that of the regulatory subunits in crystalline ATCase. Both CTP and ATP reduce rates of exchange from all seven peptides except 115-125. Although CTP slows exchange more than ATP, the effect is small except for peptides 76-91 and 78-90 which are near the nucleotide binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
Mallikarachchi
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Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06457.
Burz
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DS
Allewell
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NM
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000596:Amino Acids / Q000032:analysis; D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D003570:Cytidine Triphosphate / Q000494:pharmacology*; D003597:Cytosine Nucleotides / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008958:Models, Molecular; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation / Q000187:drug effects; D011865:Radioisotope Dilution Technique; D012996:Solutions; D014316:Tritium
D000596:Amino Acids; D003597:Cytosine Nucleotides; D046911:Macromolecular Substances; D010446:Peptide Fragments; D012996:Solutions; D014316:Tritium; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D001221:Aspartate Carbamoyltransferase