IN-SITU PROPERTIES OF HELICOBACTER-PYLORI ASPARTATE CARBAMOYLTRANSFERASE

The kinetic and regulatory properties of aspartate carbamoyltransferas e (ACTase) of the human pathogen Helicobacter pylori were studied in s itu in cell-free extracts. The presence of enzyme activity was establi shed by identifying the end product as carbamoylaspartate using nuclea r magnetic resonance spectroscopy. Activity was measured in all strain s studied, including recent clinical isolates. Substrate saturation cu rves determined employing radioactive tracer analysis or a microtiter colorimetric assay were hyperbolic for both carbamoyl phosphate and as partate, and there was no evidence for substrate inhibition at higher concentrations of either substrate. The apparent K-m were 0.6 and 11.6 mM for carbamoyl phosphate and aspartate, respectively. Optimal pH an d temperature were determined as 8.0 and 45 degrees C. Activity was ob served with the L- but not the D-isomer of aspartate. Succinate and ma leate inhibited enzyme activity competitively with respect to aspartat e. The carbamoyl phosphate analogues acetyl phosphate and phosphonoace tic acid inhibited activity in a competitive manner with respect to ca rbamoyl phosphate. With limiting carbamoyl phosphate purine and pyrimi dine nucleotides, tripolyphosphate, pyrophosphate, and orthophosphate inhibited competitively at millimolar concentrations. Ribose and ribos e 5-phosphate at 10 mM concentration showed 20 and 35% inhibition of e nzyme activity, respectively. N-Phosphonoacetyl-L-aspartate (PALA) was the most potent inhibitor studied, with 50% inhibition of enzyme acti vity observed at 0.1 mu M concentration. Inhibition by PALA was compet itive with carbamoyl phosphate (K-i = 0.245 mu M) and noncompetitive w ith aspartate. The kinetic and regulatory data on the activity of the H. pylori enzyme suggest it is a Class A ACTase, but with some interes ting characteristics distinct from this class. (C) 1997 Academic Press .