A transition state in pieces: major contributions of entropic effects to ligand binding by adenosine deaminase.
Nebularine undergoes hydration at the active site of adenosine deaminase, in a reaction analogous to a partial reaction in the displacement of ammonia from adenosine by water, to generate an inhibitory complex that captures much of the binding affinity expected of an ideal transition-state analogue. Enzyme affinities of several compounds related to nebularine 1,6-hydrate, and to its stable analog 2'-deoxycoformycin, were compared in an effort to identify the structural origins of strong binding. Binding of the stable transition-state analog inhibitor 2'-deoxycoformycin was rendered 9.8 kcal/mol less favorable by removal of substituent ribose, 9.7 kcal/mol less favorable by inversion of the 8-hydroxyl substituent of the diazepine ring, and 10.0 kcal/mol less favorable by removal of atoms 4-6 of the diazepine ring. Binding of the unstable transition-state analog nebularine hydrate was rendered at least 9.9 kcal/mol less favorable by removal of the 6-hydroxyl group and 10.2 kcal/mol less favorable by removal of atoms 1-3 of the pyrimidine ring. In each case, the enzyme exhibited only modest affinity (Kd greater than or equal to 10(-2) M) for the "missing piece", indicating that incorporation of 2 binding determinants within a single molecule permits an additional 7-12 kcal/mol of intrinsic binding energy to be manifested as observed binding energy. These results are consistent with earlier indications that adenosine deaminase may use 10.5 kcal/mol of the intrinsic free energy of binding of the two substrates to place them in positions appropriate for reaction at the active site, overcoming the unfavorable entropy change of -35 eu for the equilibrium of 1,6-hydration of purine ribonucleoside and reducing the equilibrium constant for attainment of the transition state in deamination of adenosine. Thus, adenosine deaminase may achieve up to 8 orders of magnitude of its catalytic power by converting the nonenzymatic, bimolecular, hydration reaction to a monomolecular reaction at its active site. Several new 6-substituted 1,6-dihydropurine ribonucleosides, prepared by photoaddition of formate and by low-temperature addition of organolithium reagents to a derivative of purine ribonucleoside, exhibited Ki values of 9-1400 microM against adenosine deaminase, in accord with the active site's considerable tolerance of bulky leaving groups in substrates. Inhibition by one diastereomer of 6-carboxy-1,6-dihydropurine ribonucleoside was found to be time-dependent, progressing from a weakly bound to a more strongly bound complex.
Kati
W M
WM
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599.
Acheson
S A
SA
Wolfenden
R
R
D000243:Adenosine Deaminase / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D002417:Cattle; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D008961:Models, Structural; D015394:Molecular Structure; D011485:Protein Binding; D011487:Protein Conformation; D011684:Purine Nucleosides / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D012263:Ribonucleosides / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D011684:Purine Nucleosides; D012263:Ribonucleosides; C005248:nebularine; D000243:Adenosine Deaminase