Mechanistic and structural studies of mouse adenosine deaminase


Adenosine deaminase (ADA) catalyzes the irreversible deamination of (2\sp\prime-deoxy)adenosine to (2\sp\prime-deoxy)inosine. It is an indispensable enzyme, with a role in purine catabolism and in the development of a competent immune system. This work focuses on the study of the catalytic mechanism employed by the murine enzyme through the use of site-directed mutagenesis. A glutamate mutation at the conserved active site Asp 295 shows that this residue is necessary for the proper orientation and placement of the catalytic hydroxylate. An alanine and an asparagine mutant of Asp 296 show that this residue functions mainly by anchoring the substrate in the active site via hydrogen bonding and thus reducing the aromaticity of the purine ring. Alanine, glutamate and arginine mutations at the proposed base for the reaction, His 238, clearly show that it does not abstract the proton from the zinc-bound water, but rather promotes the formation of the hydroxylate through charge stabilization. Replacements of the conserved Cys 262 by alanine and serine clearly demonstrate that it is not directly involved in the reaction mechanism. Structural studies with the ADA apoenzyme reveal that chelation of zinc does not result in structural rearrangements of either the active site or the secondary and tertiary structures of the enzyme. Loss of zinc is accompanied by loss of activity, which can be restored upon stoichiometric re-addition of zinc or cobalt. A transition-state analog such as deoxycoformycin can bind the apoenzyme by inducing the same type of conformational change as it does when it binds the holoenzyme. Mutants such as D296A and D296N denature more slowly compared to the wild-type, probably due to the better packing of an Ala or Asn side chain compared to the native Asp in the part of the enzyme surrounding residue 296. By contrast, mutants such as D295E, H238A, and H238E destabilize the holoenzyme, and mutants H238R, C262A, and C262S destabilize the holoenzyme and may also impede the in vivo folding pathway. Removal of the metal cofactor from wild-type or mutant ADA generally increases the enzyme's rate of denaturation

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This paper was published in DSpace at Rice University.

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