Transition-state discrimination by adenosine deaminase from Aspergillus oryzae

Adenosine deaminase from Aspergillus oryzae resembles mammalian adenosine deaminases in its ability to catalyze the hydrolytic removal of many substituents from C-6, and in the chirality at C-6 of the active isomer of the transition-state-analogue inhibitor 6-hydroxymethyl-1,6-dihydropurine ribonucleoside. The 5'-OH group of adenosine has been found to contribute a factor of 5.10(4) to transition-state stabilization by calf intestinal adenosine deaminase, and crystallographic observations suggest that a zinc-histidine 'bridge' is formed between the 6-OH and the 5'-OH groups of the substrate in the transition state for its deamination. The present paper describes experiments indicating that this bridge is not present during the action of adenosine deaminase from Aspergillus oryzae. We find (1), that the fungal enzyme catalyzes deamination of adenosine and 5'-deoxyadenosine with kcat/Km values that are almost identical; (2), that the Ki value of the transition-state-analogue inhibitor 2'-deoxycoformycin is much higher for the fungal enzyme (2.7.10(-9) M) than for the mammalian enzyme (2.10(-12) M) and (3), that this difference in binding affinities arises mainly from a difference in rates of enzyme-inhibitor association. Thus, the onset of inhibition was markedly slower for the fungal enzyme (kon = 1.3.10(4) M-1 s-1) than for the calf intestinal enzyme (kon = 2.6.10(6) M-1 s-1). Effects of chelating agents and divalent cations suggest that the fungal enzyme, like other deaminases for adenosine and cytidine, contains essential zinc.