Joel Van Tuttle

A comparison of the distribution and electron acceptor specificities of xanthine oxidase and aldehyde oxidase

1. 1. Tissue extracts of species from eight animal phyla were assayed for the two closely related enzymes, xanthine oxidase (E.C. 1.2.3.2) and aldehyde oxidase (E.C. 1.2.3.1). 2. 2. Species differences in the levels of aldehyde oxidase activity were much more pronounced than those of xanthine oxidase, although both enzymes were detected throughout much of the animal kingdom and were found to be mainly concentrated in liver and intestine. 3. 3. With aldehyde oxidase from most species, ferricyanide but not NAD+, was an efficient electron acceptor. 4. 4. With xanthine oxidase, three electron acceptor specificity patterns were found among the xanthine oxidases studied. 5. 5. Pattern I [NAD+>ferricyanide>O2] was found with the enzymes from the bony fishes, amphibians, reptiles and birds. 6. 6. Pattern II [ferricyanide>NAD+>O2] was common among the mammals but was also found with a few of the amphibians and reptiles studied. 7. 7. Pattern III [ferricyanide>O2>NAD+] was found only with some mammals, including man.

An enzymic synthesis of purine d-Arabinonucleosides

Abstract A method is described for the synthesis of purine d -arabinonucleosides that uses purine bases and 2,2′-anhydro-(1-β- d -arabinofuranosylcytosine), AraC-an, as the starting materials. AraC-an was chosen as the precursor to the d -arabinosyl donor, because it is more readily available than any of the products that may be sequentially derived from it, namely, 1-β- d -arabinofuranosylcytosine (AraC), 1-β- d -arabinofuranosyluracil (AraU), and α- d -arabinofuranosyl-1-phosphate (Ara f 1-P), a d -arabinofuranosyl donor. Four reactions were involved in the overall process; ( a ) AraC-an was nonenzymically hydrolyzed at alkaline pH to AraC which was then ( b ) deaminated by cytidine deaminase to AraU, a nucleoside, ( c ) phosphorylyzed by uridine phosphorylase to Ara f 1-P, and ( d ) this ester caused to react with a purine base to afford a purine d -arabinonucleoside, the reaction being catalyzed by purine nucleoside phosphorylase. All four reactions occurred in situ , the first and second being performed sequentially, whereas the third and fourth were combined in a single step. The three enzyme catalysts were purified from Escherichia coli . The efficiency of the method is exemplified by the synthesis of the d -arabinonucleosides of 2,6-diaminopurine and adenine; the overall yields, based on AraC-an, were 60 and 80%, respectively.

Xanthine oxidase activities: Evidence for two catalytically different types

Abstract Xanthine dehydrogenase (EC 1.2.1.37) from mouse small intestine was accompanied by 20% as much xanthine oxidase (EC 1.2.3.2) activity (dehydrogenase-associated oxidase). NAD + and oxygen did not compete as electron acceptors. Upon incubation at 37 °C, the dehydrogenase activity was gradually transformed to oxidase activity. Unexpectedly, the oxidase thus formed (dehydorgenase-derived oxidase) had catalytic properties different from those of the dehydrogenase-associated oxidase. The activation energy for the dehydrogenase-associated oxidase was 20,600 cal/mol, whereas that for the dehydrogenase-derived oxidase was 13,500 cal/mol. The activation energy for the dehydrogenase was 14,000 cal/mol. Between pH 6.4 and 8.5, the activity of the dehydrogenase-associated oxidase was essentially pH independent, whereas the activities of the dehydrogenase-derived oxidase and the dehydrogenase were enhanced with increasing pH. Use of the transformation inhibitor, dithiothreitol, and the protease inhibitor, diisopropylfluorophosphate, showed that these catalytic differences were not the result of partial proteolysis of the enzyme. The data demonstrate the existence of two catalytically different types of mammalian xanthine oxidase activities: A dehydrogenase-associated oxidase and a dehydrogenase-derived oxidase.

Purine Salvage Enzymes in Man and Leishmania donovani

A comparison of the enzymes of pathogenic protozoa to those of man is of fundamental importance to the search for much needed chemotherapeutic agents. The enzymes involved in purine salvage are of particular interest because most pathogenic protozoa lack the ability to synthesize purines de novo and consequently are obligate salvagers of preformed purines.

Correlation of substrate-stabilization patterns with proposed mechanisms for three nucleoside phosphorylases

Substrate-stabilization of uridine phosphorylase (uridine:orthophosphate ribosyltransferase, EC 2.4.2.3), thymidine phosphorylase (thymidine:orthophosphate deoxyribosyltransferase, EC 2.4.2.4) and purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) from Escherichia coli was investigated by heat-inactivation experiments. Nucleoside substrates stabilized uridine phosphorylase and purine-nucleoside phosphorylase, but not thymidine phosphorylase. Aglycone substrates stabilized only uridine phosphorylase. Phosphate or pentose-1-phosphate ester substrates stabilized all three enzymes. The appropriate pentose-1-phosphate ester was a more effective stabilizer than was phosphate with all three enzymes. In previous reports dealing with the kinetic analysis of these phosphorylases, sequential mechanisms were proposed. Each enzyme appeared to have different sequence of substrate addition. The substrate-stabilization patterns reported here are consistent with the proposed mechanisms.

Effects of acyclovir and its metabolites on hypoxanthine-guanine phosphoribosyltransferase

Acyclovir [9-(2-hydroxyethoxymethyl)guanine], a clinically useful anti-herpesvirus agent, was a weak inhibitor (Ki = 190 microM) of hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) from human erythrocytes. Nevertheless, this acyclic nucleoside analog was a more effective inhibitor than were its natural counterparts, guanosine (Ki = 1400 microM) and deoxyguanosine (Ki = 570 microM). The two oxidized metabolites of acyclovir, 9-carboxymethoxymethylguanine (Ki = 720 microM) and 8-hydroxy-9-(2-hydroxyethoxymethyl)guanine (Ki greater than 2000 microM), were less inhibitory than was the parent drug. None of the phosphorylated metabolites of acyclovir was as potent an inhibitor of HGPRTase as was GMP (Ki = 4 microM). However, the Ki value for acyclovir monophosphate was similar to that of dGMP (12 microM). The Ki values for acyclovir diphosphate (8.3 microM) and triphosphate (30 microM) were less than those for dGDP (110 microM) and dGTP (140 microM). The levels of these phosphate esters of acyclovir in cultured monkey kidney (Vero) and human embryo fibroblast (WI38) cells exposed to therapeutic levels of the drug were well below the observed Ki values. However, in herpesvirus-infected WI38 cells the levels of the phosphate esters of acyclovir were high enough potentially to inhibit the enzyme. Although inhibition of this enzyme by the phosphorylated metabolites of acyclovir may occur in these infected cells, concentrations of the drug very much higher than the EC50 concentration were required to achieve inhibitory levels. It is, therefore, unlikely that this inhibition contributes significantly to the antiviral activity.