PRPP-synthetase mutants

The mutant enzyme from the erythrocytes of our patient exhibited normal electrophoretic mobility. Thus, this electrophoretic method appears not to be practical for the screening of a gouty population for a PRPP synthetase mutant of this kind. On the other hand, it can not be excluded as yet that this method may show to be suitable for the detection of other mutant PRPP synthetase enzymes. 

To our knowledge the present identification of an erythrocyte PRPP synthetase mutant in the gouty family studied by us provides the first demonstration in man of excess activity of a regulatory enzyme causing an overproduction disease as a direct effect of mutation. 

The mechanism of the hyperuricemia in most individuals who have gout is unknown. Following is a discussion of biochemical lesions that lead to hyperuricemia and may eventually lead to gout. Enhanced PRPP synthesis results from X-chromosome-linked mutants of PRPP synthetase. Several variants show increased Umax, resistance to feedback inhibition, or a low for ribose 5-phosphate. 

PRPP synthetases. A. Enzyme activities in dialyzed hemolysates. Sigmoidal activation is seen for both enzymes. Activity of the mutant enzyme with an increased maximal reaction velocity is, however, increased by a constant proportion at all Pi concentrations. B. Enzyme activities in undialyzed (crude) hemolysates. The mutant enzyme, deficient in inhibitor responsiveness, shows hyperbolic activation with increased activity only at Pi concentrations below 2 mM. C. Enzyme activities in partially purified erythrocyte preparations from a normal individual and the patient with the feedback-resistant PRPP synthetase studied in B. Note hyperbolic activation of the purified normal enzyme and the resulting similarity of the Pi activation curves. D. Enzyme activities in dialyzed fibroblast extracts. The mutant enzyme, with combined increased maximal reaction velocity and diminished nucleotide responsiveness, shows both hyperbolic activation and increased enzyme activity at all Pi concentrations. 

We have recently examined the responsiveness to Mg + and MgATP of the subunit aggregates from a superactive form of PRPP synthetase with normal substrate and inhibitor binding properties but an increased maximal reaction velocity. The distribution of forms of the mutant enzyme on sucrose gradient after the dilution and incubation procedures described above was indistinguishable from that of normal PRPP synthetase. In several respects, however, the mutant enzyme behaved in an abnormal fashion. First, under conditions of suboptimal Mg2+... 

Phosphoribosylpyrophosphate (PRPP) synthetase (E.C. 2.7.6.1) catalyzes the formation of PRPP from ribose-5-phosphate and ATP in the presence of Mg and inorganic phosphate. The product, PRPP, is a substrate of the first rate limiting step of the de novo synthesis of purine nucleotides and its availability has been shown to regulate this pathway in human tissue (l). A superactive mutant erythrocyte PRPP synthetase with decreased sensitivity to feedback inhibition has recently been found by us in a gouty family (2,3). 

The results are taken to indicate the presence of only one genetic type of PRPP synthetase in the various human tissues. If valid, this conclusion would justify the conclusion that PRPP synthetase is superactive in the liver of patients with a superactive mutant erythrocyte enzyme. 

The enzyme 5-phosphoribosyl-l-pyrophosphate (PRPP) synthetase (EC 2.7.6.l) catalyzes the synthesis of PRPP from ribose-5-phosphate (R-5-P) and adenosine 5 triphosphate (ATP) in the presence of magnesium and inorganic phosphate (9-ll). In the present communication we report studies on a mutant superactive PRPP synthetase in the erythrocytes of two brothers with excessive purine production associated with gout and uric acid lithiasis. In these two patients the serum uric acid reached 13.5 and 13.6 mg percent and the urinary 24 hours uric acid excretion 2400 mg and 2250 mg, respectively. All other members of the family examined were clinically and biochemically normal, except for the mother of the patients who had hyperuricosuria, 1100 mg per 24 hours. 

DPG. In order to elucidate whether the hyperbolic response of the mutant PRPP synthetase to increasing phosphate concentration in hemolysate reflects an abnormal response to inhibitors, a system devoid of inhibitors was employed. Using stroma-free charcoal-adsorbed hemolysate treated with DEAE-cellulose, the difference in reaction to increasing inorganic phosphate concentration between the mutant enzyme and the normal enzyme disappeared both exhibiting a hyperbolic response (Fig. 2). It was furthermore found that the mutant enzyme had a decreased sensitivity to inhibition by GDP, ADP,... 

Fig. 1. Activation of normal and mutant PRPP synthetase in hemolysate by inorganic phosphate, o—o normal hemolysate ----- mutant hemolysate...

Fig. 2. Activation of partially purified normal and mutant PRPP synthetase by inorganic phosphate.

Pig. 3. Inhibition of partially purified normal and mutant PRPP synthetase enzymes by GDP, ADP, 2—3DPG and AMP. Concentration of inorganic phosphate was 1 mM. o control enzyme mutant enzyme. 

It thus appears that the mutant PRPP synthetase enzyme is structurally altered in such a way that only its regulatory properties but not its catalytic properties are affected. This selective alteration proves that these two properties are located at different sites, the enzyme being allosteric. Examples are known of mutations in bacteria (18,19) and Ehrlich ascites cells (20) which altered the susceptibility of regulatory enzymes to effector mulecules. An indication for such a mutation in man has been obtained by Henderson et al in studies on fibroblasts from two patients with purine overproduction and gout, showing reduced effectiv-ness of product inhibition of purine biosynthesis (2l). 

As expected for a biosynthetic pathway [7], the activity of the first enzyme (PR-ATP synthetase ) is controlled by the end product of the pathway [8]. At the pH used in the standard assay, pH 8.5, feedback inhibition by histidine is noncompetitive with respect to both substrates (PRPP and ATP), having a Ki of 100 fiM. When the pH is reduced to one more likely representative of that in the cell, pH 7.5, the strength of the inhibition is about doubled [8]. Feedback-resistant mutants of the PR-ATP s)mthetase have been obtained by selecting for resistance to the histidine analog 2-thiazolealanine (for structure, see Fig. 2). This analog does not substitute for histidine in proteins, but inhibits the PR-ATP synthetase [8,11,12], and thereby produces bacteriostasis in the sensitive cell by cutting off its histidine supply. Feedback-resistant mutants excrete Itistidine into the medium, whereas... 

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