Functional residues at the active site of bovine brain adenosine deaminase.
Brain adenosine deaminase was investigated in order to identify amino acid residues essential for its catalytic activity. The pH dependence of log Vmax shows that the enzyme activity depends on two ionizing groups with pK values of 5.4, that must be unprotonated, and 8.4, that must be protonated, for the catalysis. These same groups are observed in the Vmax/Km profiles. The plausible role of histidine residues at the active site of brain adenosine deaminase was proved by chemical modification with (DEP). The histidine specific reagent inactivated the enzyme following a pseudo first-order kinetics with a second-order rate constant of 8.9 10(-3) (+/- 1.8 10(-3)) M-1 min-1. The inhibition of the enzyme with PCMBS was studied monitoring the enzyme activity after incubation with the inhibitor. Brain adenosine deaminase exhibited a characteristic intrinsic tryptophan fluorescence with an emission peak centered at 335 nm. Stern-Volmer quenching parameters in the presence of acrylamide and iodide indicated that tryptophan residues are buried in the native molecule. Tryptophan residues also showed a high heterogeneity that was increased after binding of ground- and transition-state analogs to the enzyme.
Lupidi
G
G
Dept. of Biology M.C.A., University of Camerino, Italy. lupidi@cambio.unicam.it
Marmocchi
F
F
Venardi
G
G
Cristalli
G
G
D000243:Adenosine Deaminase / Q000737:chemistry / Q000378:metabolism*; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002384:Catalysis; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology; D006639:Histidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D000596:Amino Acids; D002614:Chelating Agents; D013438:Sulfhydryl Compounds; D006639:Histidine; D000243:Adenosine Deaminase; D015032:Zinc