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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.

Several adenosine analogs, such as coformycin, 2'-deoxycoformycin and erythro-9-(3-nonyl-p-aminobenzyl)adenine (EHNA), which are strong inhibitors of mammalian adenosine deaminase, are much weaker inhibitors of the Saccharomyces cerevisiae enzyme. The specificity of the yeast enzyme is more restricted than that of mammalian adenosine deaminase, particularly towards the ribose moiety and around position 6 and 1 of the substrate. The sulphydryl group appears to be more masked in the yeast than in the mammalian enzyme. The kinetic effects of pH with adenosine substrate and with the inhibitor purine riboside are reported. The findings on specificity and pH kinetic effects can be interpreted in a model involving proton transfer from the -SH group of the enzyme to the N-1 atom of the substrate.

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) from Bacillus cereus NCIB 8122 has been purified to electrophoretic homogeneity by ammonium sulfate precipitation, gel filtration through Sephadex G-100, DEAE-Sephadex A-50 chromatography and ion-exchange HPLC on DEAE-Polyol. The enzyme activity is stabilized (at temperatures from 0 degrees C to 40 degrees C) by 50 mM NH4+ or K+, while it is irreversibly lost in the absence of these or a few other monovalent cations. Glycerol (24% by volume) helps the cation in stabilizing the enzyme activity above 40 degrees C, but also exerts per se a noticeable protecting effect at room temperature. B. cereus adenosine deaminase displays the following properties: Mr on Sephadex G-200, 68,000; Mr in SDS-polyacrylamide gel electrophoresis, 53,700; optimal pH-stability (in the presence of 50 mM KCl) over the range 8-11 at 4 degrees C, and maximal catalytic activity at 30 degrees C between pH 7 and 10; Km for adenosine around 50 microM over the same pH range and Km for 2'-deoxyadenosine around 400 microM.

Two forms of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), differing in molecular size, have been purified and obtained in homogeneous form from rabbit intestine. The purification procedures involved extraction with acetate buffer, pH 5.5, precipitation and fractional reextraction with (NH4)2SO4, ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-75 and Sephadex G-200. Gel filtrations analysis gave molecular weight estimates of 265 000 and 32 000 for the large and small deaminases respectively. The two enzymes forms had similar pH optima and pH stability ranges.