End-product regulation and kinetic mechanism of guanosine-inosine kinase from Escherichia coli.
Escherichia coli guanosine-inosine kinase was overproduced, purified, and characterized. The native and subunit molecular weights were 85,000 and 45,000, respectively, indicating that the enzyme was a dimer. A pI of 6.0 was obtained by isoelectric focusing. In addition to ATP, it was found that deoxyadenosine 5'-triphosphate, UTP, and CTP could serve as phosphate donors. The phosphate acceptors were guanosine, inosine, deoxyguanosine and xanthosine, but not adenosine, cytidine, uridine, or deoxythymidine. Maximum activity was attained at an ATP/Mg2+ concentration ratio of 0.5. In the presence of pyrimidine nucleotides, enzyme activity was slightly increased, while it was markedly inhibited by GDP and GTP. Initial velocity and product inhibition studies support an ordered Bi Bi mechanism in which guanosine was the first substrate to bind and GMP was the last product to be released. Guanosine kinase may be a regulatory enzyme that has a role in modulating nucleotide levels.
Kawasaki
H
H
Applied Microbiology Laboratory, Ajinomoto Co., Inc., Kawasaki-shi, Japan. hisashi_kawasaki@ajinomoto.com
Shimaoka
M
M
Usuda
Y
Y
Utagawa
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006153:Guanosine Diphosphate / Q000096:biosynthesis / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000096:biosynthesis / Q000378:metabolism*; D007525:Isoelectric Focusing; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D004791:Enzyme Inhibitors; D009711:Nucleotides; D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C062896:inosine kinase; D008274:Magnesium