PARTIAL RECONSTITUTION OF MAMMALIAN PHOSPHORIBOSYLPYROPHOSPHATE SYNTHETASE IN ESCHERICHIA-COLI-CELLS - COEXPRESSION OF CATALYTIC SUBUNITS WITH THE 39-KDA ASSOCIATED PROTEIN LEADS TO FORMATION OF SOLUBLE MULTIMERIC COMPLEXES OF VARIOUS COMPOSITIONS

Rat liver phosphoribosylpyrophosphate (PRPP) synthetase exists as comp lex aggregates composed of 34-kDa catalytic subunits (PRS I and PRS II ) and homologous 39- and 41-kDa proteins termed PRPP synthetase-associ ated proteins (PAPs). While a negative regulatory role was indicated f or PAPs, the physiological function of PAPs is less well understood. W e attempted to prepare recombinant 39-kDa PAP (PAP39) and to reconstit ute the enzyme complex. Free PAP39 was poorly expressed in Escherichia coli, while expression of protein fused with glutathione S-transferas e was successful. The purified fusion protein had no PRPP synthetase a ctivity, and bound to dissociated PRS I and PRS II, with a similar aff inity. A free form of PAP39 prepared from the fusion protein formed in soluble aggregates. The enzyme complex was then partially reconstitute d in situ by coexpression of PAP39 with PRS I or PRS II in E. coli cel ls. This coexpression led to formation of soluble complexes of various compositions, depending on the conditions. When the relative amount o f PAP39 was higher, specific catalytic activities, in terms of the amo unt of the catalytic subunit, were lowered. PAP39 complexed with PRS I was more readily degraded by proteolysis than seen with PRS II, in vi vo and in vitro. These results provide additional, strong evidence for that PAP39 has no catalytic activity in the enzyme complex, but does exert inhibitory effects in an amount-dependent manner, and that compo sition of the enzyme complex varies, depending on the relative abundan ce of components present at the site of aggregate formation. (C) 1997 Elsevier Science B.V.