TY - JOUR
T1 - The Rate of Formation of Transition-State Analogs in the Active Site of Adenosine Deaminase is Encounter-Controlled
T2 - Implications for the Mechanism
AU - Kurz, Linda C.
AU - Moix, Luke
AU - Riley, Marian C.
AU - Frieden, Carl
PY - 1992
Y1 - 1992
N2 - 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 ~20 ps. 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.
AB - 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 ~20 ps. 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.
UR - http://www.scopus.com/inward/record.url?scp=0026499905&partnerID=8YFLogxK
U2 - 10.1021/bi00116a008
DO - 10.1021/bi00116a008
M3 - Article
C2 - 1731884
AN - SCOPUS:0026499905
SN - 0006-2960
VL - 31
SP - 39
EP - 48
JO - Biochemistry
JF - Biochemistry
IS - 1
ER -