Influence of substituent ribose on transition state affinity in reactions catalyzed by adenosine deaminase.

Adenosine deaminase from calf intestine hydrolyzes adenine at a limiting rate four orders of magnitude lower than that for adenosine, while Km values for these substrates are about the same (Wolfenden, R., et al. (1969), Biochemistry 8, 2412-2415). Reactivity of 6-substituents, toward nucleophilic displacement, is found to be affected only slightly by removal of ribose as a 9-substituent, in model reactions. Substituent ribose thus appears to stabilize, selectively, the transition state for enzymatic deamination. In contrast with the small influence of substituent ribose on the apparent binding affinity of substrates, removal of substituent ribose from a potential transition state analogue, 1,6-dihydro-6-hydroxy-methylpurine ribonucleoside, results in a lowering of its affinity for the enzyme by several orders of magnitude. The synthesis of the analogue and related compounds is described, and their properties compared with those of other photoadducts and of the naturally occurring inhibitors covidarabine and coformycin. Binding of these inhibitors is found to result in the appearance of ultraviolet-absorbing bands in the neighborhood of 323 nm.

Hydroxylation of 5,5-diphenylhydantoin (phenytoin) by dog liver microsomes.

Chang and Glazko in 1972 had reported failure to demonstrate any production from 5-5-diphenylhydantoin (phenytoin, DPH) by dog liver microsomes of either 5-(m-hydroxyphenyl-5-phenylhydantoin (m-HPPH) or 5-(P-hydroxyphenyl-5-phenylhydantoin (p-HPPH), metabolites of DPH produced by the dog in vivo. We have incubated DPH with 9,000 X g supermatants of dog liver homogenates and with suspensions of separated microsomes with added NADPH generating system, Mg(2+), and nicotinamide and have demonstrated the production of both m-HPPH and p-HPPH. Both metabolities were detected by thin-layer chromatography of extracts of the incubation mixtures. Detection and roughly quantitative measurement of m-HPPH were also accomplished with gas chromatography.

The action of bacterial cytidine deaminase on 5,6-dihydrocytidine.

Cytidine deaminase from Escherichia coli was found to catalyze the hydrolytic deamination of 5,6-dihydrocytidine, at a rate slightly lower than its rate of action on the normal substrate. The results suggest that nucleophilic addition by the enzyme at the 5,6 position of the substrate is not an essential part of catalysis, unless the active site is so flexible that deamination can occur with addition in one case (cytidine) and without addition in another case (5,6-dihydrocytidine). 3,4,5,6-Tetrahydrouridine bears a close structural resemblance to a hypothetical "tetrahedral" intermediate formed by direct water addition to 5,6-dihydrocytidine. The hydrolytic activity of the enzyme toward 5,6-dihydrocytidine and its potent inhibition by 3,4,5,6-tetrahydrouridine are presumably related by the ability of the active site to stabilize structures of this kind by tight binding. Cytidine deaminase shows no detectable activity as a catalyst for the dehydration of 6-hydroxy-5,6-dihydrouridine.

Synthesis and absolute stereochemistry of 5-alkyl-5-(3'-hydroxy-1'-methylbutyl)barbituric acid and 5-alkyl-5-(3'-hydroxy-1'-methylbutyl)-2-thiobarbituric acids.

5-Alkyl-5-(3-hydroxy-1-methylbutyl) barbituric acid (2) and 5-alkyl-5-(3-hydroxy-1-methylbutyl)-2-thiobarbituric acids (3) are matabolites of 5-alkyl-5-(2-pentyl) barbituric acid and 5-alkyl-5-(2-pantyl)-2-thiobarbituric acid, respectively. We have synthesized the four possible optical isomers of 2 and 3 by a procedure which established the absolute stereochemistry of each isomer. The two racemic pairs in each case were also prepared. The properties of these synthetic samples of 2 and 3 of known stereochemistry are compared to the properties of 2 and 3 which have been isolated from metabolism studies.