Function of threonine-55 in the carbamoyl phosphate binding site of Escherichia coli aspartate transcarbamoylase.
Carbamoyl phosphate is held in the active site of Escherichia coli aspartate transcarbamoylase by a variety of interactions with specific side chains of the enzyme. In particular, the carbonyl group of carbamoyl phosphate interacts with Thr-55, Arg-105, and His-134. Site-specific mutagenesis was used to create a mutant version of the enzyme in which Thr-55 was replaced by alanine in order to help define the role of this residue in the catalytic mechanism. The Thr-55----Ala holoenzyme exhibits a 4.7-fold reduction in maximal observed specific activity, no alteration in aspartate cooperativity, and a small reduction in carbamoyl phosphate cooperativity. The mutation also causes 14-fold and 35-fold increases in the carbamoyl phosphate and aspartate concentrations required for half the maximal observed specific activity, respectively. Circular dichroism spectroscopy has shown that saturating carbamoyl phosphate does not induce a conformational change in the Thr-55----Ala holoenzyme as it does for the wild-type holoenzyme. The kinetic properties of the Thr-55----Ala catalytic subunit are altered to a greater extent than the mutant holoenzyme. The mutant catalytic subunit cannot be saturated by either substrate under the experimental conditions. Furthermore, as opposed to the wild-type catalytic subunit, the Thr-55----Ala catalytic subunit shows cooperativity for aspartate and can be activated by N-(phosphonoacetyl)-L-aspartate in the presence of low concentrations of aspartate and high concentrations of carbamoyl phosphate. As deduced by circular dichroism spectroscopy, the conformation of the Thr-55----Ala catalytic subunit in the absence of active-site ligands is distinctly different from the wild-type catalytic subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02167.
Kantrowitz
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D000409:Alanine / Q000378:metabolism; D000970:Antineoplastic Agents / Q000378:metabolism; D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D002221:Carbamyl Phosphate / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D015394:Molecular Structure; D009154:Mutation; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D011487:Protein Conformation; D013912:Threonine / Q000378:metabolism*
D000970:Antineoplastic Agents; D013912:Threonine; D001224:Aspartic Acid; D002221:Carbamyl Phosphate; C013195:sparfosic acid; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid; D000409:Alanine