Chemical modification of essential carboxyl group and histidine residue in the plasma-membrane 5'-nucleotidase.
An investigation, using specific chemical reagents, of the amino acids involved in the catalytic activity of the purified 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) from bovine liver plasma membranes, was carried out. The enzyme was irreversibly inactivated by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The inhibition kinetics were of the first-order type and decreased partially in the presence of nucleotides and divalent cations. These results indicate for the first time that a carboxyl group is essential for the catalytic process of 5'-nucleotidase. Moreover, chemical modification by diethylpyrocarbonate also produced inactivation of the enzyme and showed a differential spectrum with a peak at 240 nm characteristic of N-carbethoxyhistidine residues. This inactivation was efficiently released upon decarbethoxylation by hydroxylamine only when the extent of inactivation, due to low concentration of diethylpyrocarbonate, was limited. The time-dependent inactivation followed first-order kinetics and nucleotides afforded significant protection against diethylpyrocarbonate modification. The results indicate the involvement of the histidine residue in catalysis.
Harb
J
J
Meflah
K
K
di Pietro
A
A
Bernard
S
S
Gautheron
D C
DC
D015720:5'-Nucleotidase; D000227:Adenine Nucleotides / Q000494:pharmacology; D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology*; D000818:Animals; D002413:Cations, Divalent; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D005561:Formates / Q000494:pharmacology*; D006639:Histidine; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009708:Nucleotidases / Q000378:metabolism*; D011804:Quinolines / Q000494:pharmacology*
D000227:Adenine Nucleotides; D000317:Adrenergic alpha-Antagonists; D002413:Cations, Divalent; D005561:Formates; D011804:Quinolines; D006639:Histidine; C000470:EEDQ; D009708:Nucleotidases; D015720:5'-Nucleotidase; D004047:Diethyl Pyrocarbonate