The rate of formation of transition-state analogues in the active site of adenosine deaminase is encounter-controlled: implications for the mechanism.
We have previously shown that purine riboside, when bound to adenosine deaminase, forms a complex in which C-6 of the purine is tetrahedral [Kurz, L. C., & Frieden, C. (1987) Biochemistry 26, 8450]. We now report the rates of formation of enzyme-inhibitor complexes of two types, those which do and those which do not form such tetrahedral intermediates. In both cases, the rates are encounter-controlled since the progress curves for formation of the complexes are well-described by a simple second-order approach to equilibrium and the rate constants show an inverse solvent viscosity dependence. Assuming that the formation of the intermediate-analogue complex is preceded by an initial ground-state analogue complex, the lifetime of that ground-state complex must be less than approximately 20 microseconds. All of the enzyme-inhibitor complexes studied share three characteristics: (1) the complexes generate large UV-difference spectra; (2) a substantial solvent isotope effect is found on the enzyme's affinity for the inhibitors; and (3) a new signal appears in the CD spectra of the complexes. Two of the nucleosides studied, 1-deazapurine riboside and 1-deaza-adenosine, form complexes which appear to mimic a ground-state rather than a reactive intermediate when bound to adenosine deaminase. We find that the values for the association rate constants for those inhibitors which form intermediate analogues are very similar to that for adenosine. The presence of a significant solvent isotope effect on the affinity of all inhibitors is attributable in part to a large transfer isotope effect on the free ligand and in part to an effect on the bound ligand. This complicates use of the solvent isotope effect in applications of the multiple isotope method for estimating intrinsic isotope effects and commitment factors.
Kurz
L C
LC
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Moix
L
L
Riley
M C
MC
Frieden
C
C
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000243:Adenosine Deaminase / Q000378:metabolism*; D058892:Adenosine Deaminase Inhibitors; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002384:Catalysis; D002417:Cattle; D002942:Circular Dichroism; D007422:Intestines / Q000201:enzymology; D011684:Purine Nucleosides / Q000378:metabolism / Q000494:pharmacology*; D012263:Ribonucleosides / Q000378:metabolism / Q000494:pharmacology*; D012997:Solvents; D013056:Spectrophotometry, Ultraviolet; D014372:Tubercidin / Q000494:pharmacology
D058892:Adenosine Deaminase Inhibitors; D011684:Purine Nucleosides; D012263:Ribonucleosides; D012997:Solvents; C048086:1-deazaadenosine; C005248:nebularine; D000243:Adenosine Deaminase; D000241:Adenosine; D014372:Tubercidin