Superactivity Of PRPP Synthetase Research Articles

Molecular Analysis of Two Enzyme Genes, HPRT1 and PRPS1, Causing X-Linked Inborn Errors of Purine Metabolism

Inherited mutation of hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch-Nyhan syndrome or HPRT-related gout. On the other hand, PRPS1 mutations cause PRPP synthetase superactivity associated with hyperuricemia and gout, sometimes including neurodevelopmental abnormalities. We have identified two mutations in two Lesch-Nyhan families after our last report. One of them, a new single nucleotide substitution (130G>T) resulting in a missense mutation D44Y was detected in exon 2 of HPRT1. RT-PCR amplification showed not only a cDNA fragment with normal size, but also a small amount of shorter fragment skipping exons 2 and 3. The other missense mutation F74L (222C > A) was detected in a Japanese patient but has been reported previously in European families. In four hyperuricemic patients with mild neurological abnormality, no mutations responsible for partial HPRT deficiency were identified in HPRT1. In these four patients, we also performed molecular analysis of PRPS1, but no mutations in PRPP synthetase were found.

Possible metabolic basis for GTP depletion in red cells of patients with PRPP synthetase superactivity.

Three different inborn enzyme abnormalities have been identified as causing excessive purine production in man [1]: deficiency of hypoxanthine guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8), superactivity of phosphoribosylpyrophosphate synthetase (PRPP synthetase; EC 2.7.6.1), and deficiency of purine nucleoside phosphorylase (PNP; EC 2.4.2.1).

Chemiluminescent assays in the study of purine metabolism.

The discovery that in cells from patients with hypoxanthine guanine phosphoribosyltransferase (HGPRT) deficiency purine overproduction is associated with an elevated intracellular concentration of phosphoribosylpyro-phosphate (PRPP) (1) has led to a reevaluation of the importance of this compound in the regulation of purine biosynthesis de novo. The work which flowed from this discovery led to the identification of an inherited super-activity of PRPP synthetase in over a dozen families in which affected individuals showed gout and/or uric acid overproduction (2). However to date HGPRT deficiency and increased activity of PRPP synthetase are the only two well documented enzyme defects of purine synthesis and reutilization identified among hyperuricemic patients. These genetic aberrations account for less 10% of all hyperuricemic patients who overproduce uric acid.

68 INCREASED PURINE NUCLEOTIDE DEGRADATION IN THE CENTRAL NERVOUS SYSTEM (CNS) IN PRPP SYNTHETASE SUPERACTIVITY

The end products of purine metabolism in the CNS are hypoxanthine for adenine nucleotides and xanthine for guanine nucleotides. Previous reports of two families with PRPP synthetase superactivity associated with neurodevelopmental defects have not documented metabolism of purines in the CNS. Fibroblasts extracts from an eight-year-old male with tophaceous gout and sensorineural deafness and from his mother with gout showed an aberrant PRPP synthetase characterized by resistance to purine nucleotide inhibition of enzyme activity. Hypoxanthine and xanthine concentrations in plasma and cerebrospinal fluid (CSF) were simultaneously measured by HPLC in both patients. In 4 normal subjects hypoxanthine and xanthine levels in plasma were (mean±SEM) 1.7±0.4 μM and 0.9±0.2 μM, respectively, and 3.3±1.1 and 2.0±0.2 μM in CSF. The hemizigous male showed a substantially increased hypoxanthine concentration of 5.6 μM in plasma and of 22.1 μM in CSF. Xanthine levels were 1.8 μM in plasma and 4.5 μM in CSF. The heterozigous female had moderately elevated plasma and CSF hypoxanthine concentrations (3.9 and 10.6 μM) and normal xanthine levels (1.3 and 1.8 μM). These results suggest an increased purine nucleotide degradation in the CNS of patients with PRPP synthetase superactivity and neurological symptoms. The predominance of hypoxanthine over xanthine may indicate a relatively enhanced adenine nucleotide over guanine nucleotide degradation.

PRPP SYNTHETASE SUPERACTIVITY IN LYMPHOBLAST LINES: 11

Superactivity of PRPP synthetase (PS) is an X chromosome-linked disorder associated with gout and uric acid overproduction. Fibroblasts from affected males show PS superactivity and increased rates of PRPP and purine nucleotide synthesis. Kinetic aberrations underlying PS superactivity include: catalytic defects; regulatory defects; substrate (ribose-5-P) binding defects; and combined catalytic and regulatory defects.We studied PS in B lymphoblasts derived from blood lymphocytes (PBL) of 6 males whose fibroblasts express the 4 classes of PS superactivity. Neither excessive PS activity nor increased rates of PRPP or purine synthesis were found in lymphoblasts from 3 patients with catalytic defects in fibroblasts, despite PS superactivity in PBL from these patients. Catalytic superactivity was also absent in lymphoblasts from a patient with combined defects in fibroblast PS. These cells and cells from 2 other patients expressed the specific regulatory and substrate binding defects found in fibroblast PS from the respective patients and also showed increased rates of PRPP and purine synthesis.Immunoreactive PS differed <20% among lymphoblast lines, and PS absolute specific activities were those of normal fibroblast PS. Thus, neither reduced levels of mutant PS nor activation of normal PS explained selective expression of PS superactivity in lymphoblasts. Altered electrophoretic mobilities and thermal stabilities, found with some catalytically superactive fibroblast PS, were absent in lymphoblasts from the respective patients. The basis of selective expression of PS superactivity remains to be determined.

INHERITED PRPP SYNTHETASE SUPERACTIVITY DUE: TO ABERRANT INHIBITOR AND ACTIVATOR RESPONSIVENESS: 12

Superactivity of PRPP synthetase (PS) is associated with purine nucleotide and uric acid overproduction, usually manifested by early adult-onset gout. We studied fibroblasts and lymphoblasts from a male child (NB) with inherited uric acid overproduction, deafness, and retarded motor development, suspected to have a superactive PS (Simmonds et al. 1982. Lancet 2:68-70). Our studies confirm a defective enzyme altered in regulation of PS activity in response to noncompetitive inhibitors and to the activator, Pi.