Adenosine deaminase converts purine riboside into an analog of a reactive intermediate: a carbon-13 NMR and kinetic study

Abstract

The 13C NMR spectra of [2-13C]- and [6-13C]purine ribosides have been obtained free in solution and bound to the active site of adenosine deaminase. The positions of the resonances of the bound ligand are shifted relative to those of the free ligand as follows: C-2, -3.7 ppm; C-6, -73.1 ppm. The binary complexes are in slow exchange with free purine riboside on the NMR time scale, and the dissociation rate constant is estimated to be 13.5 s-1 from the slow exchange broadening of the free signal. In aqueous solution, protonation of purine riboside at N-1 results in changes in 13C chemical shift relative to those of the free base as follows: C-2, -4.9 ppm; C-6, -7.9 ppm. The changes in chemical shift that occur when purine riboside binds to the enzyme indicate that the hybridization of C-6 changes from sp2 to sp3 in the binary complex with formation of a new bond to oxygen or sulfur. A change in C-2 hybridization can be eliminated as can protonation at N-1 as the sole cause of the chemical shift changes. The kinetic constants for the adenosine deaminase catalyzed hydrolysis of 6-chloro- and 6-fluoropurine riboside have been compared, and the reactivity order implies that carbon-halogen bond breaking does not occur in the rate-determining step. These observations support a mechanism for the enzyme in which formation of a tetrahedral intermediate is the most difficult chemical step. Enzymic stabilization of this intermediate may be an important catalytic strategy used by the enzyme to lower the standard free energy of the preceding transition state.

Relation of Enzyme Activity to Local/Global Stability of Murine Adenosine Deaminase: 19F NMR Studies

“…34 If the mechanism is not changed in the presence of denaturant, then subtle conformational changes in the loop b2a2 around W117 and W161, even though they are distant from the catalytic reaction center, somehow affect the formation of the tetrahedral intermediate, presumably through conformational change propagated through the structure. The naturally occurring disease-related ADA mutants around the loop b2a2 provide an indirect support of our conclusion.…”

Efficient, low-cost protein factories: expression of human adenosine deaminase in baculovirus-infected insect larvae.

“…It has been suggested that the enzyme undergoes a conformational change upon inhibitor binding (6,7) and that a reactive histidine group participates in catalysis of calf intestinal ADA (10). However, lack of sufficient quantities of human ADA has precluded detailed structural studies of important enzyme domains.…”

“…In humans deficiency of this purine salvage enzyme is associated with severe immunodeficiency and lymphopenia in both B-and T-cell lineages (1, 2 known about the enzymatic activity of human ADA, including its interaction with various substrates (4) and inhibitors (6-8), no direct information is available concerning the enzyme active site. Although a preliminary analysis of crystals of murine ADA has been reported (9), the three-dimensional fine structure of the human enzyme is not known.It has been suggested that the enzyme undergoes a conformational change upon inhibitor binding (6,7) and that a reactive histidine group participates in catalysis of calf intestinal ADA (10). However, lack of sufficient quantities of human ADA has precluded detailed structural studies of important enzyme domains.…”

“…5) of unknown function. While much is now known about the enzymatic activity of human ADA, including its interaction with various substrates (4) and inhibitors (6)(7)(8), no direct information is available concerning the enzyme active site. Although a preliminary analysis of crystals of murine ADA has been reported (9), the three-dimensional fine structure of the human enzyme is not known.…”

The Role of Zn2+on the Structure and Stability of Murine Adenosine Deaminase

“…As part of the enzymatic mechanism the Zn 2+ polarizes a water molecule, which has hydrogen bonds to His238 and Asp295, and attacks the substrate to form a tetrahedral intermediate at the C6 position of the purine ring 10–12,16. Zn 2+ binds to mADA tightly, with the dissociation constant estimated to be lower than 10 −9 M 17.…”