Contrasting behavior of conformationally locked carbocyclic nucleosides of adenosine and cytidine as substrates for deaminases

Abstract

In addition to the already known differences between adenosine deaminase (ADA) and cytidine deaminase (CDA) in terms of their tertiary structure, the sphere of Zn(+2) coordination, and their reverse stereochemical preference, we present evidence that the enzymes also differ significantly in terms of the North/South conformational preferences for their substrates and the extent to which the lack of the O(4') oxygen affects the kinetics of the enzymatic deamination of carbocyclic substrates. The carbocyclic nucleoside substrates used in this study have either a flexible cyclopentane ring or a rigid bicyclo[3.1.0]hexane scaffold.

Figure 1

Transition-state, hydrated intermediates formed during the enzymatic hydrolytic deamination of adenosine (A) and cytidine (B).

Figure 2

(A) Bovine ADA in complex with hydrated nebularine.[11] Ligand is shown in ball-and-stick representation, with green carbons. The crystal coordinates of residue His 14 are shown in dark gray. This conformation is suggested to be an average of two rotamers: one (in green) where the Nδ atom makes a bifurcated hydrogen bond to the sugar moiety in the ligand, and one (in light blue-gray) where the zinc atom lies in the plane of the imidazole ring. (B) E. coli CDA in complex with hydrated 5F-zebularine.[13]

Figure 3

Bovine spleen ADA (~0.5 units) deamination of South-dAdo (▲) and NorthdAdo (○) at 60 µM determined by measuring the decrease in absorbance at 264 nm (25 °C).

Figure 4

Full UV spectrum for the enzymatic deamination of North- and South-dCyd over time. A clean isobestic point at 250 nm is maintained throughout the course of the reaction. The inset corresponds to the drop in absorbance at 284 nm (long arrow) as a function of time. The short arrows indicate either the increase (↑) or decrease (↓) in absorbance at that point over time. A) North-dCyd (□). B) South-dCyd (♦).

Figure 5

Initial time course data for the CDA (900 nM) catalyzed deamination of North-dCyd (□) and South-dCyd (♦) followed at 284 nm (Δε²⁸⁴ = −5000). The inset shows initial time course data for the catalyzed deamination of the natural substrate cytidine (▼) by CDA (0.9 nM) monitored at 282 nm (Δε²⁸² = −3600).

Figure 6

Michaelis-Menten plot for enzyme catalyzed deamination of cytidine (▼) and carbodine (∇). The solid line represents a non-linear regression fit of the cytidine data to the Michaelis-Menten equation.