In vitro synthesis of colominic acid by membrane-bound sialyltransferase of Escherichia coli K-235. Kinetic properties of this enzyme and inhibition by CMP and other cytidine nucleotides.
The membrane-bound sialyltransferase obtained from Escherichia coli K-235 grown in a chemically defined medium (ideal for colominic acid production) was studied. The in vivo half-life calculated for this enzyme was 20 h. Kinetic tests revealed (at 33 degrees C and pH 8.3) hyperbolic behaviour with respect to CMP-Neu5Ac (Km250 microM) and a transition temperature at 31.3 degrees C. The enzyme was inhibited by NH4+, some divalent cations and by several agents that react with thiol groups. Detergents and fatty acids also inhibited the sialyltransferase activity. In vitro synthesis of colominic acid is strongly inhibited by CMP by blocking the incorporation of [14C]Neu5Ac into a protein-complex intermediate and therefore into free polymer. CDP and CTP also inhibited (91% and 84%) this enzyme activity whereas cytosine and cytidine had no effect. CMP inhibition corresponded to a competitive model the calculated Ki was 30 microM. Incubations of protein[14C]Neu5Ac with CMP, CDP and CTP led to de novo synthesis of CMP-[14C]Neu5Ac. The presence of colominic acid, which usually displaces the reaction equilibrium towards polymer synthesis, did not affect this de novo CMP-[14C]Neu5Ac formation. CMP also inhibited in vivo colominic acid biosynthesis.
Ortiz
A I
AI
Departamento de Bioquímica y Biología Molecular, Universidad de León, Spain.
Reglero
A
A
Rodríguez-Aparicio
L B
LB
Luengo
J M
JM
D002462:Cell Membrane / Q000201:enzymology; D003568:Cytidine Monophosphate / Q000494:pharmacology*; D003570:Cytidine Triphosphate / Q000494:pharmacology; D003597:Cytosine Nucleotides / Q000494:pharmacology*; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005230:Fatty Acids, Nonesterified / Q000494:pharmacology; D007700:Kinetics; D011134:Polysaccharides / Q000096:biosynthesis*; D012799:Sialyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013816:Thermodynamics
D003597:Cytosine Nucleotides; D003902:Detergents; D005230:Fatty Acids, Nonesterified; D011134:Polysaccharides; D003570:Cytidine Triphosphate; C014420:colominic acid; D012799:Sialyltransferases; D003568:Cytidine Monophosphate