A transition state in pieces: major contributions of entropic effects to ligand binding by adenosine deaminase

Abstract

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.

The Depth of Chemical Time and the Power of Enzymes as Catalysts

“…48 Earlier, adenosine deaminase was found to exhibit a similarly large connectivity effect when the enzyme's affinity for the altered substrate in the transition state, or for the transition-state analogue 1,6-dihydroinosine, was compared with those of the pieces obtained by cutting the substrate in two. 49 The magnitude of connectivity effects can be evaluated in a different way by "cutting" the enzyme, instead of the substrate, into two pieces and then comparing their catalytic activities with that of the native enzyme ( Figure 9b). In that way, one can estimate the effective concentration of that group at the enzyme's active site.…”

Synthesis of Conformationally Restricted Carbocyclic Nucleosides: The Role of the O(4′)-Atom in the Key Hydration Step of Adenosine Deaminase

“…Thus, a complete understanding of the ADA-catalyzed mechanism of deamination should provide additional means of intervening therapeutically in ADA-related disorders. The rational design of the first transition-state analogue inhibitor of ADA by Wolfenden and Evans in 1970 [6] was followed four years later by the discovery of the potent fermentation-product inhibitors coformycin (6) [7] [8] and 2'-deoxycoformycin (7) [9], which were shown to function as stable, high-affinity transition-state-analogue inhibitors of ADA [10] [11]. Further confirmation of the transition-state mimicry of these analogues came from several crystal structures of ADA with the ligands bound at the active site of the enzyme [12 ± 14].…”

“…S-type 2'-deoxy-methanocarba-adenosine 9, ((S)MCdAdo), were used as substrates for adenosine deaminase (ADA) to assess the enzymes preference for a fixed conformation relative to the flexible conformation represented by the carbocyclic nucleoside aristeromycin (10). Further comparison between the rates of deamination of these compounds with those of the two natural substrates adenosine (Ado; 1) and 2'-deoxyadenosine (dAdo; 2), as well as with that of the conformationally locked nucleoside LNA-Ado (11), which, like the natural substrates, has a furanose O(4') atom, helped differentiate between the roles of the O(4') anomeric effect and sugar conformation in controlling the rates of deamination by ADA. Differences in rates of deamination as large as 10000 can be attributed to the combined effect of the O(4') atom and the enzymes preference for an N-type conformation.…”

“…Further confirmation of the transition-state mimicry of these analogues came from several crystal structures of ADA with the ligands bound at the active site of the enzyme [12 ± 14]. In the case of the simple purine riboside 3 (nebularine), covalent hydration at the active site of ADA is able to generate the potent transition-state inhibitor (6S)-1,6-dihydro-9H-purin-6-ol riboside 3a [11] (Scheme 1). However, since the equilibrium constant for this critical hydration step is very small, the apparent inhibition constant of nebularine is only in the micromolar range [11].…”

“…In the case of the simple purine riboside 3 (nebularine), covalent hydration at the active site of ADA is able to generate the potent transition-state inhibitor (6S)-1,6-dihydro-9H-purin-6-ol riboside 3a [11] (Scheme 1). However, since the equilibrium constant for this critical hydration step is very small, the apparent inhibition constant of nebularine is only in the micromolar range [11]. On the other hand, the transition-state inhibitors coformycin (6) and 2'-deoxycoformycin (7), where the 8-hydroxy substituent of the diazepine ring provides a stable mimic of a hydrated ring, are able to inhibit ADA at picomolar levels [10] [11].…”