Kinetics of inhibition of calf intestinal adenosine deaminase by (+)- and (-)-erythro-9-(2-hydroxy-3-nonyl)adenine.
The enantiomers of erythro-9-(2-hydroxy-3-nonyl)adenine [(+)- and (-)-EHNA) bound to adenosine deaminase (ADA) at pH 7 with concomitant changes in the optical properties of the enzyme. The association rate constant for (+)-EHNA was 2.9 x 10(6) M-1 s-1 and that for (-)-EHNA was 6.4 x 10(6) M-1 s-1. The dissociation of (-)-EHNA.ADA or (+)-EHNA.ADA in the presence of excess coformycin was monitored by the quenching of enzyme fluorescence as coformycin.ADA was formed. The dissociation rate constants of (+)- and (-)-EHNA.ADA were 0.0054 s-1 and 2.7 s-1, respectively. A similar value for the dissociation rate constant (0.005 s-1) for (+)-EHNA.ADA was calculated from the time course for the appearance of catalytic activity after dilution of (+)-EHNA.ADA into 100 microM adenosine. The Ki values of ADA for (+)- and (-)-EHNA were similar to the dissociation constants calculated from the ratio of the respective dissociation and association rate constants. The biphasic time-dependent inhibition of the catalytic activity of ADA by (+/- )-EHNA [Frieden, C., Kurz, L. C., & Gilbert, H. R. (1980) Biochemistry 19, 5303-5309] was confirmed. However, the catalytic activity of ADA was inhibited monophasically by (+)-EHNA. Thus, the biphasic nature of the time course for inhibition of ADA by (+/- )-EHNA was the result of the presence of both enantiomers of the inhibitor in this assay. These kinetic data were interpreted in terms of single-step mechanisms for binding of (+)- and (-)-EHNA.
Porter
D J
DJ
Experimental Therapy Division, Wellcome Research Laboratory, Research Triangle Park, North Carolina 27709.
Abushanab
E
E
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D058892:Adenosine Deaminase Inhibitors; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D001665:Binding Sites; D002417:Cattle; D007422:Intestines / Q000201:enzymology; D007700:Kinetics; D008433:Mathematics; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013237:Stereoisomerism
D058892:Adenosine Deaminase Inhibitors; D000998:Antiviral Agents; C010192:9-(2-hydroxy-3-nonyl)adenine; D000225:Adenine