Transition-state variation in human, bovine, and Plasmodium falciparum adenosine deaminases.
Adenosine deaminases (ADAs) from human, bovine, and Plasmodium falciparum sources were analyzed by kinetic isotope effects (KIEs) and shown to have distinct but related transition states. Human adenosine deaminase (HsADA) is present in most mammalian cells and is involved in B- and T-cell development. The ADA from Plasmodium falciparum (PfADA) is essential in this purine auxotroph, and its inhibition is expected to have therapeutic effects for malaria. Therefore, ADA is of continuing interest for inhibitor design. Stable structural mimics of ADA transition states are powerful inhibitors. Here we report the transition-state structures of PfADA, HsADA, and bovine ADA (BtADA) solved using competitive kinetic isotope effects (KIE) and density functional calculations. Adenines labeled at [6-13C], [6-15N], [6-13C, 6-15N], and [1-15N] were synthesized and enzymatically coupled with [1'-14C] ribose to give isotopically labeled adenosines as ADA substrates for KIE analysis. [6-13C], [6-15N], and [1-15N]adenosines reported intrinsic KIE values of (1.010, 1.011, 1.009), (1.005, 1.005, 1.002), and (1.004, 1.001, 0.995) for PfADA, HsADA, and BtADA, respectively. The differences in intrinsic KIEs reflect structural alterations in the transition states. The [1-15N] KIEs and computational modeling results indicate that PfADA, HsADA, and BtADA adopt early SNAr transition states, where N1 protonation is partial and the bond order to the attacking hydroxyl nucleophile is nearly complete. The key structural variation among PfADA, HsADA, and BtADA transition states lies in the degree of N1 protonation with the decreased bond lengths of 1.92, 1.55, and 1.28 A, respectively. Thus, PfADA has the earliest and BtADA has the most developed transition state. This conclusion is consistent with the 20-36-fold increase of kcat in comparing PfADA with HsADA and BtADA.
Luo
Minkui
M
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Singh
Vipender
V
Taylor
Erika A
EA
Schramm
Vern L
VL
D000241:Adenosine / Q000737:chemistry; D000243:Adenosine Deaminase / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D006801:Humans; D008958:Models, Molecular; D010963:Plasmodium falciparum / Q000201:enzymology*; D011487:Protein Conformation
D000243:Adenosine Deaminase; D000241:Adenosine