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In situ properties of Helicobacter pylori aspartate carbamoyltransferase.

The kinetic and regulatory properties of aspartate carbamoyltransferase (ACTase) of the human pathogen Helicobacter pylori were studied in situ in cell-free extracts. The presence of enzyme activity was established by identifying the end product as carbamoylaspartate using nuclear magnetic resonance spectroscopy. Activity was measured in all strains studied, including recent clinical isolates. Substrate saturation curves determined employing radioactive tracer analysis or a microtiter colorimetric assay were hyperbolic for both carbamoyl phosphate and aspartate, and there was no evidence for substrate inhibition at higher concentrations of either substrate. The apparent Km were 0.6 and 11.6 mm for carbamoyl phosphate and aspartate, respectively. Optimal pH and temperature were determined as 8.0 and 45 degrees C. Activity was observed with the l- but not the d-isomer of aspartate. Succinate and maleate inhibited enzyme activity competitively with respect to aspartate. The carbamoyl phosphate analogues acetyl phosphate and phosphonoacetic acid inhibited activity in a competitive manner with respect to carbamoyl phosphate. With limiting carbamoyl phosphate purine and pyrimidine nucleotides, tripolyphosphate, pyrophosphate, and orthophosphate inhibited competitively at millimolar concentrations. Ribose and ribose 5-phosphate at 10 mm concentration showed 20 and 35% inhibition of enzyme activity, respectively. N-Phosphonoacetyl-l-aspartate (PALA) was the most potent inhibitor studied, with 50% inhibition of enzyme activity observed at 0.1 microM concentration. Inhibition by PALA was competitive with carbamoyl phosphate (Ki = 0.245 microM) and noncompetitive with aspartate. The kinetic and regulatory data on the activity of the H. pylori enzyme suggest it is a Class A ACTase, but with some interesting characteristics distinct from this class.