68 1

PURINE METABOLISM J. EDWIN SEEGhIILLER T h e symposium held at Princeton University 10 years ago provided a valuable summary of the state of knowledge of gout and purine metabolism at that time (1). T h e past decade has produced a remarkable expansion of our knowledge of the mechanism regulating purine biosynthesis i n the human (2-5). Identification of specific enzyme defects within purine metabolism responsible for these genetic defects have each in turn provided valuable information on a unifying mechanism responsible for purine overproduction ( 5 ) . I n the process, a considerable increase in understanding of tlie normal regulatory mechanism lias also emerged. Tlie development of methods for detailed study of purine metabolism i n human fibroblasts cultured from simple skin biopsies and from other cell types from normal and affected patients has contributed substantially to the rapid developments of the past decade (5-7). Tlie existence of a mechanism for synthesis of purine nucleotides de novo lias long been realized (7). T h e role of purine nucleotides i n the synthesis of DNA and RNA as well as in the formation with vitamins of a wide variety of cofactors of metabolism has made a continuous supply of purine nucleotides essential for all cells. T h e first evidence that a regulatory mechanism for control of purine synthesis is present in the human species was found in 1955 (8). T h e purine precursor 4-amino-5-imidazolecarboxamide, which requires but one carbon atom to form the purine ring, produced a marked suppression of incorporation of IjN-glycine into urinary uric acid of both normal and gouty subjects (8). Administration of adenine produced a similar suppression of purine synthesis de novo (9). Similar suppression of purine synthesis de novo had been observed in rabbit bone J . Edwiii Seegmiller, M.D.: Professor of Medicine, Department of Medicine, University of California, San Diego, La Jolla, California 92093. Address reprint requests to Dr. Seegmiller.

marrow (lo), in mouse ascites tumor cells ( l l ) , and in both mouse (1 2 ) and human fibroblasts cultured in vitro (5). Furthermore the various purine bases show a hierarchy of effectiveness in suppressing purine synthesis de novo with adenine being much more effective than hypoxanthine or guanine (1 1,13). I n most biologic systems the control of a pathway of synthesis is achieved by regulation of tlie formation of tlie first compound committed specifically to this pathway. I n the case of purine metabolism, this is the enzyme glutamine pliosplioribosylpyropliosphate amidotransferase (Figure 1). This enzyme, which was partially purified from chicken liver, was inhibited by purine 5-nucleotides formed as products of the purine biosynthetic pathway (14) and thus provided a very attractive mechanism for a continual regulation of purine synthesis to maintain the intracellular concentration of purine nucleotides at a fixed value. T h e original view of a decade ago that feedback inhibition provides the major controlling mechanism for regulation of purine biosynthesis has received little support. O n tlie contrary, considerable evidence has now accumulated for tlie operation of a n alternative mechanism for regulation of purine biosynthesis based on competition for phosphoribosylpyropliosphate (PPribose-P), the rate-limiting substrate for the presumed rate-limiting enzyme regulating purine biosynthesis, PP-ribose-P glutamine amidotransferase (Figure 2). Many of the earlier studies using whole cells which have been interpreted as evidence of feedback inhibition (11,l.Z) can now be explained i n terms of depletion of intracellular pools of PP-ribose-P (13). Tlie preferential re-utilization of purine compounds when available for synthesis of purine nucleotides with tlie consequent shutting off of purine synthesis de novo provides definite advantages for the intracellular economy of the cell. At least six A T P molecules are used during tlie step-by-step assembly of the purine ribonucleotide inosinic acid, whereas only one A T P molecule is required for formation of the same compound from

Arthritis and Rheumatism, Vol. 18, No. 6 (November-December 1975), Supplement

I

+

FH2 C

ATP

4

‘OH

fl-Phosphoribosyl-

F”2

Formyl Folic Acid Deriv.

/ j N HI

0

C

H; 0

\\

c

Formylglycinomidine ribotide (FGAM)

ribotide (FGAR)

COOH 0

I

ATP Asportic Acidc

I1

HC- NH COOH I

0

,!, f H

H2N RP

e

5-Amino-4-imidozole-N-succino-

corboxamide ribotide (S-AICR)

0

OH

II

H2N

I

C

5-Amino-4-imidazole-corboxomide ribotide !AICR)

I

CH

O=CH

I RP

RP

5-Formamido-4-imidozole-

lnosinic Acid (Hypoxonthine ribotide)

froin

Seegniiller et a1 (15) with fie! )tiis-

T h e overproduction of purines found in Xlinked uric aciduria (the LeschNylian syndrome and its variants) (22) is associated with a gross deficiency of the enzyme hypoxantliine-guanine phosphoribosyltransferase ( H P R T ) (4). It is correlated with a fourfold increase in intracellular concentration of PPribose-P (13). Less severe deficiencies of the same H P R T enzyme were found in 3 brothers who overproduced uric acid with onset of gouty arthritis relatively early in adult life (3,4,23). Two of the brothers developed renal concretions. An additional group of patients has now been defined in which the enzyme defect is not quite as severe as in children with the Lesch-Nyhan syndrome resulting in not only gouty arthritis and renal calculi, but also a variety of relatively mild attenuated neurologic disorders that include spinocerebellar syndrome, tlysartliria, spasticity, seizures, and mental retardation (3,4,23). An increased

Table 1. Relative Aflinity of PP-Ribose-P for Enzymes of Purine Metabolism (16,17)

Adenine phosphoribosyltransferase Hypoxanthirie-guanine phosphoribosyltransferase PP-ribose-P glutamine amidotransferase

,Other Purines

corboxomide ribotide (FAICAR)

hypoxanthine via the so-called “salvage” pathway. This priority system is built into the structure of the enzymes concerned with purine metabolism that utilize PP-ribose-P as shown in Table 1 (19-21). Highest priority is obviously given to re-utilization of adenine, which correlates well with the fact that adenine is a better inhibitor of purine synthesis de novo than is liypoxanthine (13).

Enzyme

CH

II

I RP

Fig 1. Enzymatic reactions involved in synthesis of purine nucleotides d e iiovo. Refiroduced sion of the publishers.

Affinity Constant (km) 33 p M

74 pM 300 pM*

T h e normal intracellular concentration of PP-ribose-P in lymphoblasts is about 10-100 p M (18). *so

I

RP

5-Ami no-4-imidorolecarboxylic acid ribotide (AICR)

*

+

N

RP

Formyl Folic Acid Deriv.

c

kP

a -N-Formylglycinomide

co2 0

5-Aminoimidozole ribotide (A1R)

o

@HN/\NH

I RP

0 II

N

NH / \ fH2 CH II

ATP Glutomine

C\ 0 0I NH

.

Glycinamide ribotide (GAR)

c6-

y-b

RP

Glycine

l-omine

AT P

NH /\

NH ,z

/NHz

H H-N-RP

fl-Phosphoribosyl-1-omine

a-Phosphoribosyl-1-pyrophosphote (PRPP)

D- Ribose-5-Phos~hote

5

682

ROLE OF HYPOXANTHINE-GUANINE PHOSPHORIBOSY LTRANSFERASE IN PURINE METABOLISM

5-Phosphoribosyl-1-Pyrophosphate (PRPP) t Glutamine

...... ..........

'.....'..b

i\oe

.**

.d'.*.'"

1

d . . . . . . . . . . . . . . . .

*-. '0.

5-P ho s p ho r i bosy I- 1 - A mine

....'.

*.

b\

\\y (r

yf,

Glycine

.*

py; k