Thomas A. Krenitsky

A comparison of the specificities of xanthine oxidase and aldehyde oxidase

Abstract This study directly compares the specificities of the structurally similar hydroxylating enzymes, aldehyde oxidase and xanthine oxidase. Michaelis-Menten constants for a variety of substrates of xanthine oxidase were in general lower than those of aldehyde oxidase. With respect to the rates of oxidation, the basic similarity was a preference for compounds having a substituted pyrimidine ring structure. Outstanding among the differences were the effects of the number and position of the ring substituents. Both enzymes readily oxidized a variety of unsubstituted and C-monosubstituted heterocycles, but only xanthine oxidase readily oxidized C-disubstituted derivatives. Certain N-substitutions, however, enhanced substrate activity with aldehyde oxidase, but markedly decreased it with xanthine oxidase. Although both enzymes preferred oxo over amino substituents, there were some specificity differences with respect to the chemical nature of substituents. Aldehyde oxidase, but not xanthine oxidase, tolerated 6-substitution of purine by alkyl, halogeno, cyano, or methylthio groups, while 6-hydroxyl or 6-methylamino substituents were tolerated only by xanthine oxidase. The position at which oxidation occurred was influenced by both the chemical nature and the positions of substituents. With some purines a different site was initially hydroxylated by each enzyme.

Amino acid ester prodrugs of acyclovir

Eighteen amino acid esters of the antiherpetic drug, acyclovir, were synthesized as potential prodrugs for oral administration. The esters were examined for in vitro antiviral activity against herpes simplex virus Type 1 (HSV-1). They were found to have less potency than the parent compound. Their efficiencies as prodrugs were evaluated in rats by measuring the urinary recovery of acyclovir. Ten prodrugs produced greater amounts of the parent drug in the urine. The L-amino acid esters were better prodrugs than the corresponding D- or D, L-isomers, suggesting the involvement of a stereoselective transporter. The L-valyl ester, 256U87, was the best prodrug. Sixty three per cent of its administered dose was excreted as acyclovir in the urine, a considerable improvement over acyclovir itself, for which this value was 19%. Since 256U87 was stable in aqueous solutions, its conversion to acyclovir in vivo was probably enzyme catalyzed. This L-valyl ester prodrug of acyclovir is now undergoing clinical evaluation.

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.

Rarity of X-Linked Partial Hypoxanthine-Guanine Phosphoribosyltransferase Deficiency in a Large Gouty Population

Abstract In a survey of 425 cases of hyperuricemia with gouty arthritis or uric acid stone, or both, we found partial deficiency of H-G PRTase, a newly recognized cause of these manifestations, in ...

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.

6-Methoxypurine arabinoside as a selective and potent inhibitor of varicella-zoster virus.

Seven 6-alkoxypurine arabinosides were synthesized and evaluated for in vitro activity against varicella-zoster virus (VZV). The simplest of the series, 6-methoxypurine arabinoside (ara-M), was the most potent, with 50% inhibitory concentrations ranging from 0.5 to 3 microM against eight strains of VZV. This activity was selective. The ability of ara-M to inhibit the growth of a variety of human cell lines was at least 30-fold less (50% effective concentration, greater than 100 microM) than its ability to inhibit the virus. Enzyme studies suggested the molecular basis for these results. Of the seven 6-alkoxypurine arabinosides, ara-M was the most efficient substrate for VZV-encoded thymidine kinase as well as the most potent antiviral agent. In contrast, it was not detectably phosphorylated by any of the three major mammalian nucleoside kinases. Upon direct comparison, ara-M was appreciably more potent against VZV than either acyclovir or adenine arabinoside (ara-A). However, in the presence of an adenosine deaminase inhibitor, the arabinosides of adenine and 6-methoxypurine were equipotent but not equally selective; the adenine congener had a much less favorable in vitro chemotherapeutic index. Again, this result correlated with data from enzyme studies in that ara-A, unlike ara-M, was a substrate for two mammalian nucleoside kinases. Unlike acyclovir and ara-A, ara-M had no appreciable activity against other viruses of the herpes group. The potency and selectivity of ara-M as an anti-VZV agent in vitro justify its further study.

Uridine phosphorylase from Escherichia coli: Kinetic properties and mechanism

Type I and Type II uridine phosphorylases (uridine: orthophosphate ribosyltransferase EC 2.4.2.3) are distinguished by their pH optima (Krenitsky et al. (1965) J. Biol. Chem. 240, 1281-1286). A Type I enzyme was partially purified from Escherichia coli. The crossing pattern of the initial velocity analysis indicated that the catalytic mechanism involved the sequential addition of substrates to the enzyme. Product inhibition by uracil or by ribose 1-phosphate was linear competitive with uridine or with concentrations of phosphate below 3 mM. This indicated that the sequence of substrate addition was random rather than ordered. At concentrations of phosphate above 3 mM, product inhibition by uracil was complex. The random mechanism of this Type I enzyme contrasts with the ordered mechanism of a Type II enzyme from rat liver (Kraut, A. and Yamada, E.W. (1971) J. Biol. Chem. 246, 2021-2030).

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.

Purification, characterization, substrate and inhibitor specificity of adenosine kinase from several Eimeria species

Ribonucleosides of some pyrazolo [3,4-d] pyrimidines have been shown to be potent anticoccidial agents. To investigate their interactions with adenosine kinase, this enzyme was purified by affinity chromatography from the sporulated oocysts of 3 avian coccidia, Eimeria tenella, E. acervulina and E. brunetti as well as from chicken liver. Comparative studies revealed several differences among the enzymes. Magnesium appeared not to be inhibitor of the E. tenella enzyme but did inhibit the enzymes from the other three sources. ATP in excess of the magnesium concentration strongly inhibited the E. brunetti enzyme but had only a small effect on the other enzymes. The chicken liver enzyme utilized a broader variety of triphosphate donors than did any of the enzymes from Eimeria species. ATP, dATP, GTP, dGTP and ITP was the best substrates. Studies with pyrazolo [3,4-d] pyrimidine nucleosides revealed two groups of enzymes with similar inhibitor specificities, the chicken liver and E. Acervulina vs. the E. tenella and E. brunetti enzyme. This grouping roughly correlates with the in vivo anticoccidial specificity of these compounds. Substrate specificity studies using two 4-substituted pyrazolo [3,4-d] pyrimidine ribonucleosides (ethylthio- and cinnamylthio-), which have shown potent anticoccidial activity in vivo, revealed that each served as a substrate for the enzymes from E. tenella and E. acervulina. The E. tenella enzyme was the more efficient at the phosphorylation of those compounds. However, only the ethylthio- compound was detectably phosphorylated by the enzyme from E. brunetti. In contrast to the inhibitor specificity, the substrate activities of these nucleosides do not correlate well with their in vivo anticoccidial activity.

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.