Studies of the quaternary structure and the chemical properties of phosphoribosylpyrophosphate synthetase from Salmonella typhimurium.

K R Schubert,R L Switzer,E Shelton

doi:10.1016/s0021-9258(19)40971-x

Abstract

Phosphoribosylpyrophosphate (PRPP) synthetase (EC 2.7.6.1) was purified to virtual homogeneity from Salmonella typhimurium cells by a modification of previously published procedures. The molecular weight of the subunit was determined to be 31,000 +/- 3,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and sedimentation equilibrium analysis of the enzyme dissolved in 6 M guanidine hydrochloride. The amino acid composition of the enzyme was determined. Proline was identified as the only NH2-terminal residue. PRPP synthetase is apparently composed of identical or nearly identical subunits. NATIVE PRPP synthetase exists in multiple states of aggregation under all conditions. However, two predominant states were demonstrated under certain conditions. A form with molecular weight of 320,000 +/- 20,000 was found at pH 7.5 in the presence of MgATP. At pH 8.2 to 8.6, with or without MgATP, the predominant form corresponded to a molecular weight of 150,000 to 200,000; sedimentation equilibrium and velocity analysis indicated 160,000 +/- 15,000 as the most reliable molecular weight. More highly aggregated forms were observed at 4 degrees and higher protein concentrations. Removal of inorganic phosphate from PRPP synthetase by dilution or dialysis resulted in disaggregation. The fundamental unit of PRPP synthetase appears to consist of five (or possibly six) subunits, which can associate to form a dimer (10 or 12 subunits) and more highly aggregated forms. A pentameric subunit structure is consistent with the multiple species resolved by electrophoresis of the native enzyme in discontinuous polyacrylamide gel systems. Visualization of PRPP synthetase by negative staining with uranyl acetate and electron microscopy revealed fields of very asymmetric molecules, the dimensions of which corresponded to the M = 160,000 form. Dimers and higher aggregates of this unit were also seen. An unusual model, in which the five subunits are asymmetrically arranged, accounts very well for the electron microscopic appearance of the enzyme. The tendency of the enzyme to aggregate is viewed as a consequence of the unsatisfied bonding regions of the fundamental asymmetric unit.

Highlights

The molecular weight of the 9.1 S species was estimated from the sedimentation coefficient and the diffusion coefficient, which was calculated from the spreading of the leading edge of the sedimenting band
We believe that these two protein species correspond to the forms of PRPP synthetase activity revealed on sucrose gradient analysis, which had sedimentation coefficients of about 10.2 S and 14.5 S as estimated by the method of Martin and Ames [31]
All of the available evidence indicates that PRPP synthetase has been isolated in nearly homogeneous form and that the enzyme is composed of subunits with a molecular weight of 31,000 + 3,000

Summary

Introduction

The molecular weight of the 9.1 S species was estimated from the sedimentation coefficient and the diffusion coefficient, which was calculated from the spreading of the leading edge of the sedimenting band. Similar determinations were made for the major sedimenting band seen at pD 8.4 and 5” after its behavior became constant These yielded a molecular weight estimate of 170,000 (D,,, = 4.9 to 5.0 x lo-’ cm set’, saO,w = 9.3 to 9.6 S). In a similar fashion an estimate of D,,, (3.9 x 10m7 cm set-‘) for the 14.3 S species was obtained from band spreading in experiments at pD 7.5 to 7.6 and 20”; a molecular weight of 330,000 was calculated for this species We believe that these two protein species correspond to the forms of PRPP synthetase activity revealed on sucrose gradient analysis, which had sedimentation coefficients of about 10.2 S and 14.5 S as estimated by the method of Martin and Ames

Methods

Results

Conclusion