William J. O'Sullivan

Alteration of quaternary structural behaviour of an hepatic orotate phosphoribosyltransferase-orotidine-5′-phosphate decarboxylase complex in rats following allopurinol therapy

Abstract The administration of allopurinol to rats was found to interfere with de novo pyrimidine biosynthesis, resulting in increased urinary excretion of orotic acid and orotidine and elevated activities of the enzymes orotate phosphoribosyltransferase and orotidine-5′-phosphate decarboxylase in erythrocytes. Similar increases in the liver enzymes were also observed, though it was necessary to dialyse the liver homogenates to observe an increase in the phosphoribosyltransferase. On the basis of thermal inactivation and gel filtration studies, evidence is presented that both enzymic activities reside in a single protein moiety capable of association-dissociation phenomena. Allopurinol administration resulted in the formation of an inhibitor (or inhibitors) of these enzymes. The inhibitor appeared to stabilize the enzymes by promoting the formation of higher molecular weight forms.

Inhibition of human erythrocyte orotidylate decarboxylase

Abstract Ribonucleotide derivatives of allopurinol and oxipurinol are potent inhibitors of human erythrocyte orotidylate decarboxylase. The inhibition constants are dependent upon the aggregation state of the enzyme, much tighter binding being observed with higher molecular weight forms. The trend was similar to that observed for K m values for orotidine-5-phosphate, the substrate of the enzyme. Of the compounds tested, 1-oxipurinol-5-phosphate, with a K i , value of 0.3 nM for the 250,000 M.W. species, was the most effective inhibitor. This was some two orders of magnitude tighter than 7-oxipurinol-5-phosphate, which in turn was two orders of magnitude tighter than 1-allopurinol-5-phosphate. A similar trend of K i estimates with molecular weight of the enzyme was observed with a number of other inhibitors, including 3-XMP, 9-XMP, 6-azaUMP, UMP and inorganic phosphate (HPO 4 2− ).

Inhibition of uridine phosphorylase from Giardia lamblia by pyrimidine analogs

Fifty-six pyrimidine analogs were tested as possible inhibitors of uridine phosphorylase from Giardia lamblia. Values of Ki were determined for eight of these which demonstrated an inhibition greater than 60% under the standard conditions of uridine at 1 mM (approximately 1.5 times the Km) and inhibitor at 1 mM. All were competitive with respect to uridine. The most effective inhibitors were uracil analogs substituted at the C-5 position with electron withdrawing groups (nitro groups or halogens). The inhibitory effect at the 5-position appeared to be further enhanced by substitution at the C-6 position with electron releasing groups. The order of effectiveness as inhibitors was 6-methyl-5-nitrouracil greater than 6-amino-5-nitrouracil greater than 5-benzylacyclouridine greater than 5-nitrouracil greater than 5-fluorouracil greater than 5-bromouracil greater than 6-benzyl-2-thiouracil greater than 1,3-dimethyluracil with Ki values of 10, 12, 44, 56, 119, 230, 190 and greater than 1000 microM, respectively. The compounds were also effective inhibitors of the thymidine phosphorylase activity of the enzyme. The effect of the more potent compounds on G. lamblia in in vitro culture are currently under investigation.

The orotic aciduria of pregnancy

Abstract A mild orotic aciduria is found to be associated with human pregnancy. The excretion of orotic acid was found to be at approximately the same level as observed for heterozygotes of hereditary orotic aciduria. While considerably greater than normal, it is doubtful if the excess excretion poses any threat to the developing fetus. No pattern in orotic acid excretion during gestation was observed, nor were there any significant changes in the erythrocyte enzymes, orotate phosphoribosyltransferase and orotidine-5′-monophosphate decarboxylase, which convert orotic acid to uridine-5′-monophosphate.

Purification and characterization of uracil phosphoribosyltransferase from Crithidia luciliae.

1. Uracil phosphoribosyltransferase (UPRTase) was purified 370-fold from the protozoan parasite, Crithidia luciliae. 2. The enzyme was a dimer of mol. wt 80 000 and was highly specific for uracil. 3. GTP, which is an activator of UPRTase from E. coli had a slight inhibitory effect on the parasite enzyme. 4. The C. luciliae UPRTase demonstrated a broad specificity for activating divalent metal ions.

Binding of MnADP− to phosphoglycerate kinase

7 ~ • Sources and ~he enzyme s reactmn kinetics •have been re;,dewe d recendy by Scopes r ] ]. Investigations have dezut subst~r~tialIy with !;he Mnetic .activation of the enzyme by metal ion z of actors [2 -5 ] 7 l~e!imina.ry X-ray cryst.allcgrat)h~e studies taave been reported on the enzyme from yeast [6] and ~ o r s e snusc] .e [ 7 ] R e c e n f l T ~ a 3 . 5 A r e s o l u t i o n N e c tron density map of the yeast enzymeTwas C ~ c u l a t e d [8]. ;By soaking e~,,stals in sol, tion~ containing magnesium to as and ADP, the. position of a unique MgADP (or.MnADP) binding site on One lobe o f the enzyme waslocated, though no definite conclusions about the mode o.f binding ofthe metal ionwere ieached [8]

Multiple molecular forms of orotidylate decarboxylase from human liver

Abstract Orotidylate decarboxylase activity from human liver has been demonstrated to be associated with a number of molecular weight species. Depending on ionic strength, the presence of thiols and storage condition of the liver, variable proportions of forms with molecular weights of 35,000, 63,000, and 105,000 could be separated on Sephadex G-150. Fresh liver, which had been frozen at −70°, gave predominantly the 63,000 form, which was considered to be the most likely form to occur in vivo . This form could be dissociated to the 35,000 form by low ionic strength and aggregated to a much higher MW form in the absence of thiols. The dissociation could be reversed by incubation with UMP. Orotate phosphoribosyltransferase co-eluted from Sephadex G-150 with the 63,000 form of the decarboxylase. Its activity was much more labile than the decarboxylase and was completely lost during any interconversions.

Allopurinol and enzymes of de novo pyrimidine biosynthesis

Abstract Since its introduction in the treatment of hyperuricemia, allopurinol, an inhibitor of purine biosynthesis (1), has also been shown to interfere with de novo pyrimidine biosynthesis (2–4). In particular, coordinately increased levels of the enzymes orotidylate phosphoribosyltransferase (OPRTase) and orotidine-5′-monophosphate decarboxylase (ODCase) have been observed in the erythrocytes of patients on allopurinol therapy (2–5), accompanied by increased excretion of orotic acid and orotidine. To account for these findings it has been proposed that in vivo formation of oxipurinol (the principal metabolite of allopurinol) ribonucleotide, catalyzed by OPRTase after allopurinol administration, leads to inhibition of ODCase (5). In view of the coordinate increase in activity of both ODCase and OPRTase [presumably coded by structural genes constituting a single operon (6)], it was important to ascertain whether the activity of other enzymes of de novo pyrimidine biosynthesis were affected by allopurinol. It has been shown that in cultured human fibroblasts, inhibitors of uridine-5′-monophosphate (UMP) synthesis lead to increased activity of both OPRTase and ODCase, but not of the two preceding enzymes in the pyrimidine biosynthetic pathway (dihydroorotase and dihydroorotate dehydrogenase) (7), and a similar finding would be expected with allopurinol in vivo , were an allopurinol metabolite acting as an ODCase inhibitor as proposed. The following study presents results on the effect of allopurinol therapy on the activities of OPRTase, ODCase and the first specific enzyme of de novo pyrimidine biosynthesis, aspartate transcarbamylase (ATCase), in human erythrocytes and rat liver. The effect of allopurinol upon pyrimidine metabolism, as a function of duration of therapy, in two normal adult males is also presented.

Studies on manganese-substrate complexes of arginine kinase from Panulirus longipes

Abstract Measurements of the relaxation rate of water protons in the presence of Mn2+ were used to demonstrate the formation of a ternary MnADP-enzyme complex with arginine kinase (EC 2.7.3.3) from Panulirus longipes. The enhancement of the proton relaxation rate of the ternary complex was determined to be 18 and the dissociation constant of MnADP− from the complex as 30 μM. An independent estimate of the dissociation constant for MnADP− of 20 μM, was obtained by initial velocity measurements of the enzymic reaction. Evidence for the formation of ternary complexes with the enzyme was also obtained for the manganous complexes of deoxy ADP, IDP, GDP, ATP, deoxy ATP and ITP. The enhancement of the proton relaxation rate was significantly decreased on the addition of l -arginine to MnADP-enzyme, or Mn-deoxy ADP-enzyme, indicative of the formation of a quaternary dead-end complex, enzyme-metal-substrate- l -arginine.