Bp. Burns et al., IN-SITU PROPERTIES OF HELICOBACTER-PYLORI ASPARTATE CARBAMOYLTRANSFERASE, Archives of biochemistry and biophysics, 347(1), 1997, pp. 119-125
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
.